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United States Patent 6,022,708
de Sauvage ,   et al. February 8, 2000
Fused

Abstract

The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, vectors and host cells expressing, immunoadhesins, agonists and antagonists to human & vertebrate fused.

Inventors: de Sauvage; Frederic (Foster City, CA); Rosenthal; Arnon (Burlingame, CA)
Assignee: Genentech, Inc. (South San Francisco, CA)
Appl. No.: 031563
Filed: February 26, 1998

Current U.S. Class: 435/69.1; 435/252.3; 435/252.33; 435/254.2; 435/254.21; 435/320.1; 435/325; 435/358; 536/23.5
Intern'l Class: C12P 021/02; C12N 001/21; C12N 005/10; C12N 015/63
Field of Search: 435/69.1,70.1,70.3,71.1,71.2,252.3,252.33,254.11,254.2,254.21,320.1,325,358 536/23.1,23.2,23.5

References Cited
U.S. Patent Documents
5602171Feb., 1997Tang et al.514/455.
5710173Jan., 1998Tang et al.514/447.
Foreign Patent Documents
WO 96/35124Nov., 1996WO.
WO 96/40276Dec., 1996WO.


Other References

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Johnson et al., "Human Homolog of Patched, a Candidate Gene for the Basal Cell Nevus Syndrome" Science 272:1668-1671 (1996).
Krauss et al., "A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos" Cell 75:1431-1444 (1993).
Krishnan et al., "Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase" Science 278(5345):1947-1950 (Dec. 12, 1997).
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Marti et al., "Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants" Nature 375(6529):322-325 (May 25, 1995).
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Pham et al., "The Suppressor of fused gene encodes a novel Pest protein involved in Drosophila segment polarity establishment" Genetics 140(2):587-598 (Jun. 1995).
Preat, "Characterization of Suppressor of fused, a complete suppressor of the fused segment polarity gene of Drosophila melanogaster" Genetics 132(3):725-736 (Nov. 1992).
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Unden et al., "Mutations in the human homologue of Drosophila patched (PTCH) in basal cell carcinomas and the Gorlin syndrome: different in vivo mechanisms of PTCH inactivation" Cancer Research 56(20):4562-4565 (Oct. 15, 1996).
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van den Heuvel and Ingham, "Smoothened Encodes a Receptor-Like Serpentine Protein Required for Hedgehog Signalling" Nature 382:547-551 (1996).
Wicking et al., "Most germ-line mutations in the nevoid basal cell carcinoma syndrome lead to a premature termination of the Patched protein, and no genotype-phenotype correlations are evident" American Journal of Human Genetics 60(1):21-26 (Jan. 1997).
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Zamecnik et al., "Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA" Proc. Natl. Acad. Sci. 83:4143-4146 (1986).
Myers (1996) Human sequence tagged site SHGC-32400. GenBank accession No. G27519. GenBank record date Jun. 28, 1996. Accessed Nov. 9, 1998.

Primary Examiner: Degen; Nancy
Assistant Examiner: Schwartzman; Robert
Attorney, Agent or Firm: Svoboda; Craig G.
Claims


What is claimed is:

1. Isolated nucleic acid comprising DNA selected from the group consisting of:

(a) a nucleotide sequence encoding the sequence of amino acids 1 to about 260 of FIG. 1 (SEQ ID NO:24);

(b) a nucleotide sequence comprising at least 780 residues which hybridizes under stringent conditions to the complement of the nucleotide sequence encoding SEQ ID NO:24;

(c) a nucleotide sequence which encodes SEQ ID NO:24 with at least one conservatively substituted amino acid residue; and

(d) a nucleotide sequence which encodes SEQ ID NO:24 with an addition or deletion of one to five amino acid residues.

2. The isolated nucleic acid of claim 1 comprising DNA encoding the sequence of amino acids 1 to 1315 (SEQ ID NO:2) of FIG. 1.

3. The isolated nucleic acid of claim 1 comprising DNA encoding a polypeptide having a lysine at amino acid position 33 of SEQ ID NO:24.

4. The isolated nucleic acid of claim 1 comprising a nucleotide sequence encoding the sequence of amino acids 1 to about 260 of FIG. 1 (SEQ ID NO:24).

5. The isolated nucleic acid of claim 1 comprising a nucleotide sequence comprising at least 780 residues which hybridizes under stringent conditions to the complement of the nucleotide sequence encoding SEQ ID NO:24.

6. The isolated nucleic acid of claim 1 comprising a nucleotide sequence which encodes SEQ ID NO:24 with at least one conservatively substituted amino acid residue.

7. The isolated nucleic acid of claim 1 comprising a nucleotide sequence which encodes SEQ ID NO:24 with an addition or deletion of one to five amino acid residues.

8. A vector comprising the nucleic acid of claim 1.

9. The vector of claim 8 operably linked to control sequences recognized by a host cell transformed with the vector.

10. A host cell transformed with the vector of claim 9.

11. The host cell of claim 10 which is mammalian.

12. The host cell of claim 10 which is prokaryotic.

13. The host cell of claim 10 wherein said cell is a yeast cell.

14. A process for producing a polypeptide comprising culturing the host cell of claim 10 under conditions suitable for expression of a polypeptide encoded by the nucleic acid of claim 1 and recovering the polypeptide from the cell culture.

15. The host cell of claim 11 wherein said cell is a CHO cell.

16. The host cell of claim 12 wherein said cell is an E. coli.

17. The host cell of claim 13 which is Saccharomyces cerevisiae.

18. An isolated nucleic acid comprising DNA of at least 780 residues which (a) hybridizes under stringent conditions to the complement of the nucleotide sequence encoding the cDNA in ATCC Deposit No. 209637, or (b) the complement of the DNA of (a).

19. An isolated nucleic acid comprising DNA encoding (a) the same mature polypeptide encoded by the cDNA in ATCC Deposit No. 209637 with at least one conservative substitution, or (b) the complement of the DNA or (a).

20. An isolated nucleic acid comprising DNA encoding (a) the same mature polypeptide encoded by the cDNA in ATCC Deposit No. 209637 with an addition or deletion of one to five amino acid residues, or (b) the complement of the DNA of (a).

21. An isolated nucleic acid comprising DNA selected from the group consisting of:

(a) a nucleotide sequence which encodes a human fused polypeptide comprising the sequence of amino acids 1 to 1315 of FIG. 1 (SEQ ID NO:2);

(b) a nucleotide sequence comprising at least 780 residues which hybridizes under stringent conditions to the complement of the nucleotide sequence encoding SEQ ID NO:2;

(c) a nucleotide sequence which encodes SEQ ID NO:2 with at least one conservatively substituted amino acid residue; and

(d) a nucleotide sequence which encodes SEQ ID NO:2 with an addition or deletion of one to five amino acid residues.

22. The isolated nucleic acid of claim 21 comprising a nucleotide sequence which encodes a human fused polypeptide comprising the sequence of amino acids 1 to 1315 of FIG. 1 (SEQ ID NO:2).

23. The isolated nucleic acid of claim 21 comprising a nucleotide sequence of at least 780 residues which hybridizes under stringent conditions to the complement of the nucleotide sequence encoding SEQ ID NO:2.

24. The isolated nucleic acid of claim 21 comprising a nucleotide sequence which encodes SEQ ID NO:2 with at least one conservatively substituted amino acid residue.

25. The isolated nucleic acid of claim 21 comprising a nucleotide sequence which encodes SEQ ID NO:2 with an addition or deletion of one to five amino acid residues.
Description


FIELD OF THE INVENTION

The present invention relates generally to signaling molecules, specifically to signaling and mediator molecules in the hedgehog (Hh) cascade which are involved in cell proliferation and differentiation.

BACKGROUND OF THE INVENTION

Development of multicellular organisms depends, at least in part, on mechanisms which specify, direct or maintain positional information to pattern cells, tissues, or organs. Various secreted signaling molecules, such as members of the transforming growth factor-beta (TGF-.beta.), Wnt, fibroblast growth factors and hedgehog families have been associated with patterning activity of different cells and structures in Drosophila as well as in vertebrates. Perrimon, Cell: 80: 517-520 (1995).

Hedgehog (Hh) was first identified as a segment-polarity gene by a genetic screen in Drosophila melanogaster, Nusslein-Volhard et al, Roux. Arch. Dev. Biol. 193: 267-282 (1984), that plays a wide variety of developmental functions. Perrimon, supra. Although only one Drosophila Hh gene has been identified, three mammalian Hh homologues have been isolated: Sonic Hh (SHh), Desert Hh (DHh) and Indian Hh (IHh), Echelard et al, Cell 75: 1417-30 (1993); Riddle et al, Cell 75: 1401-16 (1993). SHh is expressed at high level in the notochord and floor plate of developing vertebrate embryos. In vitro explant assays as well as ectopic expression of SHh in transgenic animals show that SHh plays a key role in neuronal tube patterning, Echelard et al., supra., Krauss et al., Cell 75, 1431-44 (1993), Riddle et al, Cell 75: 1401-16 (1993), Roelink et al, Cell 81: 445-55 (1995). In vitro explant assays as well as ectopic expression of SHh in transgenic animals show that SHh plays a key role in neural tube patterning, Echelard et al. (1993), supra.; Ericson et al., Cell 81: 747-56 (1995); Marti et al, Nature 375: 322-5 (1995); Roelink et al. (1995), supra; Hynes et al, Neuron 19: 15-26 (1997). Hh also plays a role in the development of limbs (Krauss et al, Cell 75: 1431-44 (1993); Laufer et al, Cell 79, 993-1003 (1994)), somites (Fan and Tessier-Lavigne, Cell 79 1175-86 (1994); Johnson et al, Cell 79: 1165-73 (1994)), lungs (Bellusci et al., Develop. 124: 53-63 (1997) and skin (Oro et al, Science 276: 817-21 (1997). Likewise, IHh and DHh are involved in bone, gut and germinal cell development, Apelqvist et al., Curr. Biol 7: 801-4 (1997); Bellusci et al., Development 124: 53-63 (1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al., Development 121: 3163-74 (1995). SHh knockout mice further strengthened the notion that SHh is critical to many aspect of vertebrate development, Chiang et al., Nature 383: 407-13 (1996). These mice show defects in midline structures such as the notochord and the floor plate, absence of ventral cell types in neural tube, absence of distal limb structures, cyclopia, and absence of the spinal column and most of the ribs.

At the cell surface, the Hh signals is thought to be relayed by the 12 transmembrane domain protein Patched (Ptch) [Hooper and Scott, Cell 59: 751-65 (1989); Nakano et al., Nature 341: 508-13 (1989)] and the G-protein coupled like receptor Smoothened (Smo) [Alcedo et al., Cell 86: 221-232 (1996); van den Heuvel and Ingham, Nature 382: 547-551 (1996)]. Both genetic and biochemical evidence support a receptor model where Ptch and Smo are part of a multicomponent receptor complex, Chen and Struhl, Cell 87: 553-63 (1996); Marigo et al., Nature 384: 176-9 (1996); Stone et al., Nature 384: 129-34 (1996). Upon binding of Hh to Ptch, the normal inhibitory effect of Ptch on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. Loss of function mutations in the Ptch gene have been identified in patients with the basal cell nevus syndrome (BCNS), a hereditary disease characterized by multiple basal cell carcinomas (BCCs). Disfunctional Ptch gene mutations have also been associated with a large percentage of sporadic basal cell carcinoma tumors, Chidambaram et al., Cancer Research 56: 4599-601 (1996); Gailani et al., Nature Genet. 14: 78-81 (1996); Hahn et al., Cell 85: 841-51 (1996); Johnson et al., Science 272: 1668-71 (1996); Unden et al., Cancer Res. 56: 4562-5 (1996); Wicking et al., Am. J. Hum. Genet. 60: 21-6 (1997). Loss of Ptch function is thought to cause an uncontrolled Smo signaling in basal cell carcinoma. Similarly, activating Smo mutations have been identified in sporatic BCC tumors (Xie et al., Nature 391: 90-2 (1998)), emphasizing the role of Smo as the signaling subunit in the receptor complex for SHh. However, the exact mechanism by which Ptch controls Smo activity still has yet to be clarified and the signaling mechanisms by which the Hh signal is transmitted from the receptor to downstream targets also remain to be elucidated. Genetic epistatic analysis in Drosophila has identified several segment-polarity genes which appear to function as components of the Hh signal transduction pathway, Ingham, Curr. Opin. Genet. Dev. 5: 492-8 (1995); Perrimon, supra. These include a kinesin-like molecule, Costal-2 (Cos-2) [Robbins et al., Cell 90: 225-34 (1997); Sisson et al, Cell 90: 235-45 (1997)], a protein designated fused [Preat et al., Genetics 135: 1047-62 (1993); Therond et al., Proc. Natl Acad Sci. USA 93: 4224-8 (1996)], a novel molecule with unknown function designated Suppressor offused [Pham et al., Genetics 140: 587-98 (1995); Preat, Genetics 132: 725-36 (1992)] and a zinc finger protein Ci. [Alexandre et al., Genes Dev. 10: 2003-13 (1996); Dominguez et al., Science 272: 1621-5 (1996); Orenic et al, Genes Dev. 4: 1053-67 (1990)]. Additional elements implicated in Hh signaling include the transcription factor CBP [Akimaru et al., Nature 386: 735-738 (1997)], the negative regulator slimb [Jiang and Struhl, Nature 391: 493-496 (1998)] and the SHh response element COUP-TFII [Krishnan et al., Science 278: 1947-1950 (1997)].

Mutants in Cos-2 are embryonicly lethal and display a phenotype similar to Hh over expression, including duplications of the central component of each segment and expansion domain of Hh responsive genes. In contrast, mutant embryos for fused and Ci show a phenotype similar to Hh loss of function including deletion of the posterior part of each segment and replacement of a mirror-like image duplication of the anterior part or each segment and replacement of a mirror-like duplication of the anterior part, Busson et al., Roux. Arch. Dev. Biol. 197: 221-230 (1988). Molecular characterizations of Ci suggested that it is a transcription factor which directly activates Hh responsive genes such as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez et al., (1996) supra. Likewise, molecular analysis offused reveals that it is structurally related to serine threonine kinases and that both intact N-terminal kinase domain and a C-terminal regulatory region are required for its proper function, Preat et al., Nature 347: 87-9 (1990); Robbins et al., (1997), supra; Therond et al., Proc. Natl. Acad. Sci. USA 93: 4224-8 (1996). Consistent with the putative opposing functions of Cos-2 andffused,fused mutations are suppressed by Cos-2 mutants and also by Suppressor of fused mutants, Preat et al., Genetics 135: 1047-62 (1993). However, whereas fused null mutations and N-terminal kinase domain mutations can be fully suppressed by Suppressor of fused mutations, C-terminus mutations of fused display a strong Cos-2 phenotype in a Suppressor of fused background. This suggests that the fused kinase domain can act as a constitutive activator of SHh signaling when Suppressor of Fused is not present. Recent studies have shown that the 92 kDa Drosophila fused, Cos-2 and Ci are present in a microtubule associated multiprotein complex and that Hh signaling leads to dissociation of this complex from microtubules, Robbins et al, Cell 90: 225-34 (1997); Sisson et al., Cell 90: 235-45 (1997). Both _fused and Cos-2 become phosphorylated in response to Hh treatment, Robbins et al., supra; Therond et al., Genetics 142: 1181-98 (1996), but the kinase(s) responsible for this activity(ies) remain to be characterized. To date, the only known vertebrate homologues for these components are members of the Gli protein family (e.g., Gli-1, Gli-2 and Gli-3). These are zinc finger putative transcription factors that are structurally related to Ci. Among these, Gli-1 was shown to be a candidate mediator of the SHh signal [Hynes et al., Neuron 15: 35-44 (1995), Lee et al., Development 124: 2537-52 (1997); Alexandre et al., Genes Dev. 10: 2003-13 (1996)] suggesting that the mechanism of gene activation in response to Hh may be conserved between fly and vertebrates. To determine whether other signaling components in the Hh cascade are evolutionarily conserved and to examine the function of fused in the Hh signaling cascade on the biochemical level, Applicants have isolated and characterized the human fused cDNA. Tissue distribution on the mouse indicates that fused is expressed in SHh responsive tissues. Biochemical studies demonstrate that fused is a functional kinase. Functional studies provide evidence that fused is an activator of Gli and that a dominant negative form of fused is capable of blocking SHh signaling in Xenopus embryos. Together this data demonstrated that fused is directly involved in Hh signaling.

Applicants have identified a cDNA encoding a human fused (hfused) polypeptide and thus have provided for the first time a vertebrate fused molecule.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encoding a fused polypeptide comprising the sequence of amino acids 1 to 260 of FIG. 1 (SEQ ID NO:24), or (b) the complement of the DNA molecule of (a); and encoding a polypeptide having fused biological activity. The sequence identity preferably is about 85%, more preferably about 90%, most preferably about 95%. In one aspect, the isolated nucleic acid has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with a polypeptide having amino acid residues 1 to about 1315 of FIG. 1 (SEQ ID NO:2). Preferably, the highest degree of sequence identity occurs within the kinase domain (amino acids 1 to about 260 of FIG. 1 (SEQ ID NO:2)). Especially preferred are those nucleic acid molecule containing a coding sequence for a lysine at amino acid position 33. In a further aspect, the isolated nucleic acid molecule comprises DNA encoding a human fused polypeptide having amino acid residues 1 to about 260 of (SEQ ID NO:2 as shown in FIG. 1. In yet another aspect, the invention provides for an isolated nucleic acid comprising DNA having at least a 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the cDNA in ATCC Deposit No. 209637 (designation: pRK5tkneo.hFused-1272), alternatively the coding sequence of clone pRK5tkneo.hFused-1272, deposited under accession number ATCC 209637. In a still further aspect, the invention provides for a nucleic acid comprising human fused encoding sequence of the cDNA in ATCC deposit No. 209637 (designation: pRK5tkneo.hFused-1272) or a sequence which hybridizes thereto under stringent conditions.

In another embodiment, the invention provides a vector comprising DNA encoding a vertebrateflsed polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be mammalian cells, (e.g. CHO cells), prokaryotic cells (e.g., E. coli) or yeast cells (e.g., Saccharomyces cerevisiae). A process for producing vertebrate fused polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of vertebratefused and recovering the same from the cell culture.

In yet another embodiment, the invention provides an isolated vertebrate fised polypeptide. In particular, the invention provides isolated native sequence vertebrate fused polypeptide, which in one embodiment is a human fused including an amino acid sequence comprising residues 1 to about 1315 of (SEQ ID NO:2) as shown in FIG. 1. Human and other native vertebrate fused polypeptides with or without the initiating methionine are specifically included. Alternatively, the invention provides a vertebrate fused polypeptide encoded by the nucleic acid deposited under accession number ATCC 209637.

In yet another embodiment, the invention provides chimeric molecules comprising a vertebrate fused polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a vertebrate fused polypeptide fused to an epitope tag sequence or a constant region of an immunoglobulin.

In yet another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide sequences identified in FIG. 2 as 2515662 (SEQ ID NO:3).

In yet another embodiment, the invention provides for compounds and methods for developing antagonists against and agonist promoting fused modulation of Hedgehog signaling. In particular, an antagonist of vertebrate fused which blocks, prevents, inhibits and/or neutralized the normal functioning of fused in SH signaling pathway, including both small bioorganic molecules and antisense nucleotides.

In yet another embodiment, the invention provides for alternatively spliced variants of human fuised. In still yet a further embodiment, the invention provides a method of screening or assaying for identifying molecules that modulate the fused activation of hedgehog signaling. Preferably, the molecules either prevent interaction offused with its associative complexing proteins or prevent or inhibit dissociation of complexes. The assay comprises the incubation of a mixture comprising fused and a substrate (e.g., Gli, COUP-TFII, slimb, CBP, MBP) with a candidate molecule and detection of the ability of the candidate molecule to modulate fused phosphorylation of its substrate. The screened molecules preferably are small molecule drug candidates. In particular, the method relates to a technique for screening for antagonists or agonists of fused biological activity comprising:

(a) exposing the fused expressing target cells in culture to a candidate compound; and

(b) analyzing cell lysates to asses the level and/or identity of phosphorylation; or

(c) scoring phenotypic or functional changes in treated cells; and comparing the results to control cells which were not exposed to the candidate compound. In yet another embodiment, the method relates to a technique of diagnosing to determine whether a particular disorder is modulated by hedgehog signaling, comprising:

(a) culturing test cells or tissues;

(b) administering a compound which can inhibitfitsed modulated hedgehog signaling; and

(c) measuring the degree of kinase attenuation on the fised substrate in cell lysates or hedgehog mediated phenotypic effects in the test cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show the nucleotide (SEQ ID NO:1) and derived amino acid (SEQ ID NO:2) sequence of a native sequence of human fused polypeptide. Included are the kinase domain (residues 1 to about 260 (SEQ ID NO:24)) and the ATP binding site at about amino acid position 33.

FIG. 2 shows the EST 2515662 (SEQ ID NO:3) that was used in the cloning of the human full-length fused sequence.

FIGS. 3A-3E show a comparison between human and Drosophila fused (SEQ ID NOS: 2 and 23, respectively). Gaps introduced for optimal alignment are indicated by dashes. Identical amino acids are boxed. The lysine residue mutated infused-DN (dominant negative, lysine at amino acid position 33) is shown with a star.

FIGS. 4A-4F show the sequence of DNA28495 (SEQ ID NOS:4, 5 and 21) that was an incorrectly spliced variant of human fuised isolated from a fetal lung library. This clone contains a potential initiation methionine at position 116 followed by an open reading frame of 1944 bp. A second open reading frame is present from about position 2295 to 4349. There is one nucleotide difference between clone DNA28495 (SEQ ID NO:4) and clone DNA28494 (SEQ ID NO:6) located in the first ORF at position 1863 of clone DNA28495 (SEQ ID NO:4) (A vs. G) which changes the coding sequence from an Gln to a Arg at position 583. The first open reading frame of DNA28494 (SEQ ID NO:6) starts at residue 115 and is followed by a 630 amino acid long open reading frame.

FIGS. 5A-5F show the sequence of DNA28494 (SEQ ID NOS:6, 7 and 22) that was another incorrectly spliced variant of human fused isolated from a fetal lung library.

FIG. 6 is a western blot of the PCR product of an epitope tag of DNA 28495 (SEQ ID NO:5 and 21) and DNA28494 (SEQ ID NOS:5 and 22). A specific band of 150 kDa was detected in the cell pellet of cells transfected with the construct corresponding to clone DNA28494 (SEQ ID NO:6) and a specific band of approximately 100 kDa could be detected for clone DNA28495 (SEQ ID NO:4) (FIG. 6). These bands were not present in the mock transfected control. The presence of the 100 kDa band suggests the two open reading frames of DNA28494 (SEQ ID NO:6) can be spliced together to direct the synthesis of a large protein of 150 kDa. The absence of this band for DNA28495 (SEQ ID NO:4) suggested that this clone apparently cannot be correctly spliced.

FIG. 7 is a northern blot analysis of human fused (SEQ ID NO: 1). Multiple human fetal and adult tissue northern blots were probes with a human fused (SEQ ID NO:1) cDNA probe.

FIGS. 8A-8F are photographs showing in situ hybridization of embryonic and adult tissues with fused (SEQ ID NO:1). Sagittal sections of E11.5 (FIG. 8A) and E13.5 (FIG. 8B) mouse embryos. Coronal section through the spinal chord of E11.5 (FIG. 8C) and E13.5 (FIG. 8D) mouse embryo. Sagittal section through P1 (FIG. 8E) and adult (FIG. 8F) mouse. Cp, choroid plexus; hb, hindbrain; hip, hippocampal formation; ht, heart; hy, hypothalamus; kd, kidney; lg, lung; mb, midbrain; md, midgut; mnd, mandibular component of first branchial arch; sc, spinal cord; st, stomach; tec, midbrain tectum; vh, ventral horn of spinal cord; vm, ventral midbrain. Scale bars: FIG. 8A, 1.0 mm; FIG. 8B, 1.62 mm; FIG. 8C, 0.14 mm; FIG. 8D, 0.17 mm; FIG. 8E, 2.0 mm; FIG. 8F, 3.1 mm.

FIGS. 9A-9C are photographs showing in situ hybridization showing the presence of fused mRNA in high levels in the adult mouse testes. (FIG. 9A) High magnification reveals differences in levels of expression within seminiferous tubules (FIG. 9C). Hybridization of the testis with a sense strand control probe to fused gave no hybridization (FIG. 9B).

Figures 10A-10B are bar graphs representing the activation of Gli by fused. (FIG. 10A) C3H10T1/2 cells were cotransfected with a p9XGliLus, ptkRenilla luciferase and fused or various fused mutants. Cells were harvested 48h after transfection and the luciferase activity was assayed as described in Example 7. (FIG. 10B) Fused transactivation of a Gli reporter construct. C3H10T1/2 cells were cotransfected with a p9XGliLuc reporter construct, ptkRenilla luciferase and a CMV driven expression vector for fused or various fused mutants. Cells were harvested 48 hours after transfection and the luciferase activity was assayed as described in the Examples. The data represents the mean of duplicative determinations.

FIGS. 11A-11E are photographs showing that fused-DN (SEQ ID NO:25) inhibits SHh signaling in early Xenopus development. Depicted are: (FIG. 11A) Dorsal view of tadpole stage embryos. Top embryo is fused-DN (SEQ ID NO. 25) injection and bottom embryo is the control; (FIG. 11B) Side view of tadpole stage embryo. Top embryo is fused-DN injection and bottom embryo is the control; (FIGS. 11C & 11D) Pax-6 staining of stage 16 neurula embryos injected with control DNA and fused-DN (SEQ ID NO:25), respectively; (FIG. 11E) SHh expression in the floor plate of neurula stage control embryo (left) or fused-DN injected embryo (right).

FIG. 12 is a photograph which confirms the kinase activity of fused (SEQ ID NO:2) and its activation of Gli. Depicted are 293 cells transfected with HA tagged fiised constructs as indicated in Example 10 and immunoprecipitated with anti-HA antibodies and protein A sepharose. Protein A beads were subjected to in vitro kinase assay as described in Example 10 in the presence of MBP.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms "vertebrate fitsed" and "vertebrate fused polypeptide" when used herein encompass native sequence vertebrate fused and vertebrate fused variants (which are further defined herein) having fused biological activity. Fused may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.

A "native sequence vertebratefitsed" comprises a polypeptide having the same amino acid sequence as a vertebrate fused derived from nature. Such native sequence vertebrate fused can be isolated from nature or can be produced by recombinant and/or synthetic means. The term "native sequence vertebrate fused" specifically encompasses naturally occurring truncated forms of vertebrate fused , naturally occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of vertebrate fused. Native vertebrate fused includes e.g., fused in mammals such as human, murine, bovine, porcine, equine, feline, canine, etc., and preferably refers to human. Thus, one embodiment of the invention, the native sequence human vertebrate fused is a mature or full-length native human vertebrate fused comprising amino acids 1 to 1315 of (SEQ ID NO:2) as shown in FIG. 1 with or without the initiating methionine at position 1.

"Vertebrate fused variant" means an active vertebrate fused as defined below having at least about 80% amino acid sequence identity to (a) a DNA molecule encoding a vertebrate fused polypeptide, or (b) the complement of the DNA molecule of (a). In a particular embodiment, the vertebrate fused variant has at least about 80% amino acid sequence homology with the vertebrate fused having the deduced amino acid sequence (SEQ ID NO:2) shown in FIG. 1 for a full-length native sequence vertebrate fused. Such vertebrate fused variants include, without limitation, vertebrate fused polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the sequence of FIG. 1 (SEQ ID NO:2). Preferably, the nucleic acid or amino acid sequence identity is at least about 85%, more preferably at least about 90%, and even more preferably at least about 95%.

"Percent (%) amino acid sequence identity" with respect to the vertebrate fused sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the vertebrate fused sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

"Percent (%) nucleic acid sequence identity" with respect to the vertebrate fused sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the vertebrate fused sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising vertebrate fused polypeptide, or a portion thereof, fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the vertebrate fused polypeptide. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 to about 50 amino acid residues (preferably, between about 10 to about 20 residues).

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesin comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesins may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2, IgE, IgD or IgM. Immunoadhesion reported in the literature include fusions of the T cell receptor* [Gascoigne et al., Proc. Natl. Acad. Sci USA 84: 2936-2940 (1987)]; CD4* [Capron et al, Nature 337: 525-531 (1989); Traunecker et al., Nature 339: 68-70 (1989); Zettrneissl et al., DNA Cell Biol. USA 9: 347-353 (1990); Byrn et al., Nature 344, 667-670 (1990)]; L-selectin (homing receptor) [Watson et al., J. Cell. Biol. 110, 2221-2229 (1990); Watson et al., Nature 349, 164-167(1991)]; CD44* [Aruffo et al., Cell 61, 1303-1313 (1990)];CD28* and B7* [Linsley et. al., J Exp. Med. 173, 721-730(1991)];CTLA-4* [Lisley et al., J. Exp. Med. 174, 561-569(1991)]; CD22* [Stamenkovic et al., Cell 66. 1133-1144(1991)]; TNF receptor [Ashkenazi et al., Proc. Natl. Acad Sci. USA 88, 10535-10539 (1991); Lesslauer et al., Eur. J. Iimunol 27, 2883-2886(1991); Peppel et al., J Exp. Med. 174, 1483-1489(1991)]; NP receptors [Bennett et al., J. Biol. Chem. 266, 23060-23067 (1991)]; IgE receptor .alpha.-chain* [Ridgway and Gorman, J. Cell. Biol. 115, abstr. 1448 (1991)]; HGF receptor [Mark, M.R. et al., 1992, submitted], where the asterisk (*) indicates that the receptor is member of the immunoglobulin superfamily.

"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends upon the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their T.sup.m (melting temperature). The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. Moreover, stringency is also inversely proportional to salt concentrations. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology (1995).

"Stringent conditions," as defined herein may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.015 M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpurrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; (3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide at 55.degree. C., followed by a high-stringency wash consisting of 0.1.times.SSC containing EDTA at 55.degree. C.

"Moderately stringent conditions" may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), and include the use of a washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than described above. An example of moderately stringent conditions is a condition such as overnight incubation at 37.degree. C. in a solution comprising: 20% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1.times.SSC at about 37-50.degree. C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the vertebrate fused natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

An "isolated" vertebrate fused nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the vertebrate fused nucleic acid. An isolated vertebratefuised nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated vertebrate fused nucleic acid molecules therefore are distinguished from the corresponding native vertebratefuised nucleic acid molecule as it exists in natural cells.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyeptopic specificity, as well as antibody fragments (e.g., Fab, F(ab').sub.2 and Fv), so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1 975), or may be made by recombinant DNA methods [see, e.g. U.S. Pat. No. 4,816,567 (Cabilly et al.)].

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity [U.S. Pat. No. 4,816,567; Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855 (1984)].

"Humanized" forms of non-human (e.g. murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, corresponding non-human residues replace Fv framework residues of the human immunoglobulin. Furthermore, humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see: Jones et al., Nature 321, 522-525 (1986); Reichmann et al., Nature 332, 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2 593-596 (1992) and U.S. Pat. No. 5,225,539 (Winter) issued Jul. 6, 1993.

"Active" or "activity" for the purposes herein refers to form(s) of vertebrate fused which retain the biologic and/or immunologic activities of native or naturally occurring vertebrate fused. A preferred activity is the ability to bind to and affect, e.g., block or otherwise modulate, hedgehog signaling. The activity preferably involves the regulation of the pathogenesis of Basal cell carcinoma. Another preferred biological activity is the ability to phosphorylate or modulate the phosphorylation of Gli.

The term "antagonist" is used herein in the broadest sense to include any molecule which blocks, prevents, inhibits, neutralizes the normal functioning offitsed in the Hh signaling pathway. One particular form of antagonist includes a molecule that interferes with the interaction between fused and its binding or complexing proteins. In a similar manner, the term "agonist " is used herein to include any molecule which promotes, enhances or stimulates the normal functioning offiused in the Hh signaling pathway. Suitable molecules that affect the protein-protein interaction of fused and its binding proteins include fragments of the latter or small bioorganic molecules, e.g., peptidomimetics, which will prevent or enhance, as the case may be, the interaction of proper complex formation. Non-limiting examples include proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Another preferred form of antagonist includes antisense nucleotides that inhibit proper transcription of wild type fused. Preferred forms of antagonists and are small molecules, which specifically bind to or block binding of the ATP binding site of fused.

The term "modulation" or "modulating" means upregulation or downregulation of a signaling pathway. Cellular processes under the control of signal transduction may include, but are not limited to, transcription of specific genes; normal cellular functions, such as metabolism, proliferation, differentiation, adhesion, apoptosis and survival, as well as abnormal processes, such as transformation, blocking of differentiation and metastasis.

The techniques of "polymerase chain reaction," or "PCR", as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and/or DNA are amplified as described in U.S. Pat. No. 4,683,195 issued Jul. 28, 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primer will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR sequences form total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Qziant. Biol. 51: 263 (1987); Erlich, Ed., PCR Technology, (Stockton Press, N.Y., 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid test sample comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.

II. Compositions and Methods of the Invention

A. Full-length vertebrate fused

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as human and vertebrate jiused. In particular, Applicants have identified and isolated cDNA encoding a vertebrate fused polypeptide, as disclosed in further detail in the Examples below. Using BLAST, BLAST-2 and FastA sequence alignment computer programs, Applicants found that a full-length native sequence human fised (shown in FIG. 3 (SEQ ID NO:2)) has 28% amino acid sequence identity with Drosophila fissed (SEQ ID NO:23). Accordingly, it is presently believed that the human fused disclosed in the present application is a newly identified member of the hedgehog signaling cascade.

The full-length native sequence of human vertebrate Jiused gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length gene or to isolate still other vertebrate homolog genes (for instance, those encoding naturally-occurring variants of vertebrate jused or vertebrate fused from other species) which have a desired sequence identity to the vertebrate fused sequence disclosed in FIG. 1 (SEQ ID NO:1). Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from the nucleotide sequence of FIG. 1 (SEQ ID NO:1) or from genomic sequences including promoters, enhancer elements and introns of native sequence vertebrate fused. By way of example, a screening method will comprise isolating the coding region of the vertebrate fused gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as .sup.32 P or .sup.35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the vertebrate fused gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to.

B. Vertebrate fused Variants

In addition to the full-length native sequence vertebrate fused described herein, it is contemplated that vertebrate fused variants can be prepared. Vertebrate fused variants can be prepared by introducing appropriate nucleotide changes into a known vertebrate fused DNA, or by synthesis of the desired vertebrate fused polypeptides. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the vertebrate fused.

Variations in the native full-length sequence vertebrate fused or in various domains of the vertebrate fused described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the vertebrate fused that results in a change in the amino acid sequence of the vertebrate fused as compared with the native sequence vertebrate fused. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the vertebrate fused. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the vertebrate fused with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity in the in vitro assay described in the Examples below.

The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the vertebrate fused variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main- chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J Mol. Biol, 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

In the human fused sequence depicted in FIG. 1, the kinase domain is represented by amino acid residues 1-260 (SEQ ID NO:24) of which position lysine 33 appears to be necessary for ATP binding and thus enzymatic activity.

C. Modifications of vertebrate fused

Covalent modifications of vertebrate fused are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of the vertebrate fised with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the vertebrate fused. Derivatization with bifunctional agents is useful, for instance, for crosslinking vertebrate fused to a water-insoluble support matrix or surface for use in the method for purifying anti-vertebrate fused antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1 -bis(diazo-acetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, for example, esters with 4-azido-salicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis-(succinimidyl-propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)-dithio]pro-pioimi-date.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha.-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of vertebrate fused comprises linking the vertebrate fused polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Such modifications would be expected in increase the half-life of the molecules in circulation in a mammalian system; Extended half-life of fused molecules might be useful under certain circumstances, such as where the fused variant is administered as a therapeutic agent.

The vertebrate fused of the present invention may also be modified in a way to form a chimeric molecule comprising vertebrate fused bonded to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of the vertebrate fused with a tag polypeptide, which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the vertebrate fused. The presence of such epitope-tagged forms of the vertebrate fused can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the vertebrate fused to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of the vertebrate fused with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule. Ordinarily, the C-terminus of a contiguous amino acid sequence of a ligand- (IFN-.gamma.-) binding domain of an IFN-.gamma. receptor is fused to the N-terminus of a contiguous amino acid sequence of an immunoglobulin constant region, in place of the variable region(s), however N-terminal fusions are also possible.

Typically, such fusions retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHl of the heavy chain or the corresponding region of the light chain. This ordinarily is accomplished by constructing the appropriate DNA sequence and expressing it in recombinant cell culture. Alternatively,immunoadhesinsmay be synthesized according to known methods.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of the immunoadhesins.

In a preferred embodiment, the C-terminus of a contiguous amino acid sequence which comprises the binding site(s) for IFN-y is fused , at the N-terminal end, to the C-terminal portion of an antibody (in particularthe Fc domain), containing the effector functions of an immunoglobulin, e.g. immunoglobulin G.sub.1 (IgG-1). As hereinabove mentioned, it is possible to fuse the entire heavy chain constant region to the sequence containing the binding site(s). However, more preferably, a sequence beginning in the hinge regionjust upstream of the papain cleavage site (which defines IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 114 [Kobet et al., supra], or analogous sites of other immunoglobulins) is used in the fusion. Although it was earlier thought that in immunoadhesins the immunoglobulin light chain would be required for efficient secretion of the heterologous protein-heavy chain fusion proteins, it has been found that even the immunoadhesins containing the whole IgG1 heavy chain are efficiently secreted in the absence of light chain. Since the light chain is unnecessary, the immunoglobulinheavy chain constant domain sequence used in the construction of the immunoadhesins of the present invention may be devoid of a light chain binding site. This can be achieved by removing or sufficiently altering immunoglobulin heavy chain sequence elements to which the light chain is ordinarily linked so that such binding is no longer possible. Thus, the CH1 domain can be entirely removed in certain embodiments of the IFN-.gamma. receptor-immunoglobulinchimeras.

In a particularly preferred embodiment, the amino acid sequence containing the extracellular domain of an IFN-.gamma. receptor is fused to the hinge region and CH2, CH3; or CH1, hinge, CH2 and CH3 domains of an IgG-1, IgG-2, IgG-3, or IgG-4 heavy chain. The construction of a typical structure is disclosed in Example 1.

In some embodiments, the IFN-.gamma. receptor-immunoglobulin molecules (immunoadhesins) are assembled as monomers, dimers or multimers, and particularly as dimers or tetramers. Generally, these assembled immunoadhesins will have known unit structures similar to those of the corresponding immunoglobulins. A basic four chain structural unit (a dimer of two immunoglobulin heavy chain-light chain pairs) is the form in which IgG, IgA and IgE exist. A four chain unit is repeated in the high molecular weight immunoglobulins; IgM generally exists as a pentamer of basic four-chain units held together by disulfide bonds. IgA globulin, and occasionally IgG -globulin, may also exist in a multimeric form in serum. In the case ofmultimers, each four chain unit may be the same or different.

It is not necessary that the entire immunoglobulin portion of the IFN-.gamma. receptor-immunoglobulinchimeras be from the same immunoglobulin. Various portions of different immunoglobulins may be combined, and variants and derivatives of native immunoglobulins can be made as hereinabove described with respect to IFN-.gamma., in order to optimize the properties of the immunoadhesin molecules. For example, immunoadhesin constructs in which the hinge of Igl-1 was replaced with that of IgG-3 were found to be functional and showed pharmacokinetics comparable to those of immunoadhesinscomprisingthe entire IgG-1 heavy chain.

Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol, 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Enoineering 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., Bio Technology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)]. A preferred tag is the influenza HA tag.

D. Preparation of vertebrate fused

The description below relates primarily to production of a particular vertebrate fused by culturing cells transformed or transfected with a vector containing vertebrate fused nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare vertebrate fused. For instance, the vertebrate fused sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of the vertebrate fused may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length vertebrate fused.

1. Isolation of DNA Encoding vertebrate closed

DNA encoding vertebrate fused may be obtained from a cDNA library prepared from tissue believed to possess the vertebrate fused mRNA and to express it at a detectable level. Accordingly, human vertebrate fused DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The vertebrate fused-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the vertebrate fused or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding vertebrate fused is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like .sup.32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer softwvare programs such as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT which employ various algorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloning vectors described herein for vertebrate fused production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, for example, CaPO.sub.4 and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterizim tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriae such as E. coli. Various E. coli strains are publicly available, such as E. coli K 12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vertebrate fused-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.

Suitable host cells for the expression of vertebrate fused are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol,, 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad Sci USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cell is deemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding vertebrate fused may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques, which are known to the skilled artisan.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. A preferred replicable expression vector is the plasmid is pRK5. Holmes et al., Science, 253:1278-1280 (1991).

Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the vertebrate fused nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operably linked to the vertebrate fused nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the .beta.-lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res. 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding vertebrate fused.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem, 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

Vertebrate fused transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

Inserting an enhancer sequence into the vector may increase transcription of a DNA encoding the vertebrate fused by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the vertebrate fused coding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding vertebrate fused.

Still other methods, vectors, and host cells suitable for adaptation to the synthesis of vertebrate fused in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Exiression

Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence vertebrate fused polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to vertebrate fused DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of vertebrate fused may be recovered from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of vertebrate fused can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify vertebrate fiused from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the vertebrate fused. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzvmoloey, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular vertebrate fused produced.

E. Uses for vertebrate fused

(1) Fuised is universal mediator of Hh signaling

The human fused full length molecule of human (FIG. 1 (SEQ ID NO: 1)) encodes a protein with a predicted molecular weight of 150 kDa, which is significantly larger that Drosophila fused (100 kDa, dfused (SEQ ID NO:23)). Human fused (hfused) shows notable homology to the Drosophila homologue in the kinase domain, but little homology with dfused or any other known protein over the remaining .apprxeq.1000 amino acids. The kinase domain extends from residue 1 to about residue 260, as is represented in FIG. 1 (SEQ ID NOS:24 and 2). This divergence at the C-terminus of the molecules is unexpected given that the C-terminus of the Drosophila molecule is required for its activity, Preat et al., Nature 347: 87-9 (1990). An ATP binding site is at about amino acid position 33 and is required for kinase activity.

Prior studies in Drosophila indicate that dfused is necessary for Hh signal to occur but have not addressed the issue whether fused is sufficient to activate this signaling system. As depicted in the Examples, applicants have herein used a Gli DNA binding element present in the HNF3.beta. promoter, in front of a luciferase mediator of the Hh cascade, which clearly demonstrates that fused alone is capable of activating Gli mediated transcription in this system. It is further apparent that both an intact kinase domain and an intact C-terminal non-catalytic domain are required for this activation, which supports the notion that fused functions as a kinase and that the C-terminus may play a role in the substrate recognition or in regulating the kinase activity.

Applicants have shown in the present application that hfused is a kinase which is capable of phosphorylating artificial substrates such as MBP. However, the identity of the physiological substrate for hfused remains to be determined. One obvious candidate is Gli-1 itself, as Gli-1 phosphorylation by hfiosed can be detected in vitro.

To determine if human fused is essential for Hh signaling in vertebrates, a mutant was constructed by altering a conserved lysine in the ATP binding site (about amino acid residue 33). Typically, such mutants act as inhibitor of the corresponding wild type kinase by blocking access to substrate and/or regulatory factors, He et al., Nature 374, 617-22 (1995). When overexpressed in 2-cell stage Xenopus embryos, the most remarkable phenotype was the presence of fused eyes in about 30% of the injected embryos. Several lines of evidence indicate that this phenotype is likely to result from the inhibition of Hh signaling. First, SHh knockouts display a cyclopia phenotype attributed recently to mutations in the SHh gene, Chiang et al., Nature 383: 407-13 (1996). Second, zebrafish embryos (cyclops) with reduced expression of SHh or injected with constitutively active form of PKA, a negative regulator of the Hh pathway are cyclops. Third, SHh, emanating from prechordal plate, has been shown to inhibit expression of Pax-6, a key transcription factor required for eye development, in the center of a continuous eyefield, Ekker et al., Curr. Biol. 5: 944-55 (1995); Li et al., Development. 124: 603-15 (1997); Macdonald et al., Development 121: 3267-78 (1995). Finally, staining for Pax-6 embryos injected with fused -DN revealed a single field of expression suggesting a failure of SHh emanating from the prechordal plate to downregulate the expression of Pax-6 at the center of the eyefield.

To confirm the position of fused in the Hh signaling pathway, expression of SHh in the floor plate of Xenopus embryos injected with hfitsed-DN could be rescued by coinjection of Gli-1. This suggests that fused acts in association with Gli in the SHh signaling pathway.

The tissue distribution of fused shows that it is expressed in all SHh responsive cells. In particular, its expression pattern overlaps well with Ptch, the binding component of the Hh receptor which is itself a target gene of the SHh signaling pathway. These data suggest that fused is involved in mediating a wide variety of effect SHh has on different tissues. Functionally, this was observed again in frog embryos where, fused-DN inhibited eye development as well as SHh expression in the floor plate.

hFused-DN also appears to affect normal development of tissues such as the frog gut which is regulated by Indian Hh. This, combined with the fact that fused is expressed in the gut and testis, sites of IHh and DHh action respectively, suggest that fuised may be a universal mediator of signaling for all members of the Hh protein family.

Very high levels of fused mRNA was found on germ cell, the development of which appears to be regulated by DHh. Homozygous mutant mice for DHh fail to develop germ cells and are viable but sterile (Bitgood et al., Curr. Biol. 6: 298-304 (1996). However, Patched, a Hedgehog receptor is expressed on interstitial Leydig cells and not on germ cells where fused is expressed, Bitgood et al, supra. This discrepancy suggests that there may be additional hedgehog receptors.

Applicants have shown in the Examples that wild type h fused is capable of activating Gli in a reporter assay. Furthermore, expression of SHh in the floor plate of frog embryos injected with hfused-DN could be rescued by coinjection of Gli-1. Taken together these observations are consistent with the assertion that fused acts downstream of Smo and upstream of Gli in this signaling pathway, which is consistent with the genetic evidence in Drosophila to date.

(2) General usesfor vertebrate fused

Nucleotide sequences (or their complement) encoding vertebrate fused have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. Vertebrate fused nucleic acid will also be useful for the preparation of vertebrate fused polypeptides by the recombinant techniques described herein.

The full-length native sequence vertebrate fused gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of vertebrate fused or vertebrate fused from other species) which have a desired sequence identity to the vertebrate fused sequence disclosed in FIG. 1 (SEQ ID NO:1). Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from the nucleotide sequence of FIG. 1 or from genomic sequences including promoters, enhancer elements and introns of native sequence vertebrate fused. By way of example, a screening method will comprise isolating the coding region of the vertebrate fused gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as .sup.32 p or .sup.35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the vertebrate fused gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine to which members of such libraries the probe hybridizes. Hybridization techniques are described in further detail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related vertebrate fused sequences.

Nucleotide sequences encoding a vertebrate fused can also be used to construct hybridization probes for mapping the gene, which encodes vertebrate fused and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.

Vertebrate fused polypeptides can be used in assays to identify the other proteins or molecules involved in complexing with fused which ultimately results in the modulation of hedgehog signaling. Alternatively, the these molecules can modulate the fused kinase phosphorylation of its substrate. By such methods, inhibitors of the binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the substrate of vertebrate fused can be used to isolate correlative complexing proteins. Screening assays can be designed to find lead compounds that mimic the biological activity of a native vertebrate fused or to find those that act as a substrate for vertebrate fused. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Such small molecule inhibitors could block the enzymatic action of fused , and thereby inhibit hedgehog signaling. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.

Nucleic acids which encode vertebrate fused or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA that is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding vertebrate fused can be used to clone genomic DNA encoding vertebrate fused in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding vertebrate fused. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for vertebrate fused transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding vertebrate fused introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding vertebrate fused. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. For example, for basal cell carcinoma, fused can be overexpressed in the basal cell layer of the skin using a Keratin 5 or 14 promoter. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Non-human homologues of vertebrate fused can be used to construct a vertebrate fised "knock out" animal which has a defective or altered gene encoding vertebrate fused as a result of homologous recombination between the endogenous gene encoding vertebrate fused and altered genomic DNA encoding vertebrate fused introduced into an embryonic cell of the animal. For example, cDNA encoding vertebrate fused can be used to clone genomic DNA encoding vertebrate fused in accordance with established techniques. A portion of the genomic DNA encoding vertebrate fused can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the vertebrate fused polypeptide.

As fused has been implicated as a universal mediator for all member of the Hh family (SHh, IHh, DHh), disease states or disorders which are associated with general Hh signaling, would also be treatable with fused and antagonists and agonists thereof. For example, SHh activation (e.g. fused agonists) has recently been promoted as a treatment for various degenerative disorders of the nervous system, e.g., Parkinson's disease, memory deficits, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, schizophrenia, stroke and drug addiction. Recent studies suggest that Dhh mutant males are infertile due to the failure of spermatocytes to complete their differentiation into mature sperm, Bitgood et al., Curr. Biol. 6: 298-304 (1996); Bitgood et al., Dev. Biol. 172:126-138 (1995). Additionally, fused agonists could be used to great gut diseases, bone diseases, skin diseases, diseases of the testis, ulcers, lung diseases, diseases of the pancreas, diabetes, osteoporosis.

The presence of the protein kinase domain suggests that fused may act similarly as members of the protein kinase family in the modulation of Hh signaling. Protein kinases are essential elements of regulatory circuits in differentiated as well as growing cells; Preat et al., Nature 347: 87-89 (1990). Many of these enzyme are involved in transduction of extracellular signals and operate through a cascade of phosphorylation events that amplify and disseminate the effects of a primary signal. As described earlier, Drosophila fused bears significant homology to other intracellular serine/threonine kinases. Many serine/threonine kinases are implicated in cell-cycle control in yeasts and in mammals, Hunter, Cell 50: 823-829 (1987); Dunphy & Newport, Cell 55: 925-928 (1988); Lee & Nurse, Trend. Genet. 4: 287-290 (1988).

Suppression or inhibition of Hh signaling is also an objective of therapeutic strategies. Since inactive fused has been shown to inhibit Hh signaling, it follows that a fused antagonist would also be expected to be antagonistic to Hh signaling. Limiting Hh signaling would be useful in disease states or disorders characterized by Hh signaling. For example, SHh is known to be active in Basal Cell Carcinoma; DHh is known to be active in spermatogenesis. Inhibitor or antagonist of Hh signaling would be effective therapeutics in the treatment of Basal Cell Carcinoma or male contraception, respectively.

The stimulation of Hh signaling is also an objective of therapeutic strategies. Activating Hh signaling would be useful in disease states or disorders characterized by inactive or insufficient Hh signaling. For example, degenerative disorders of the nervous system, e.g., Parkinson's disease, memory deficits, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, schizophrenia, stroke and drug addiction. Additionally, fused agonists could be used to great gut diseases, bone diseases, skin diseases, diseases of the testis (including infertility), ulcers, lung diseases, diseases of the pancreas, diabetes, osteoporosis.

F. Anti-vertebrate fitsed Antibodies

The present invention further provides anti- vertebrate fused antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-vertebrate fised antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the vertebrate fused polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-vertebrate fused antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the vertebrate fused polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the un fused , immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against vertebrate fused. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

3. Humanized Antibodies

The anti-vertebrate fused antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1 534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)].

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the vertebrate fused, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

G. Uses for anti-vertebrate fused Antibodies

The anti-vertebrate fused antibodies of the invention have various utilities. For example, anti-vertebrate fused antibodies may be used in diagnostic assays for vertebrate fused, eg., detecting its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as .sup.3 H, .sup.14 C, .sup.32 P, .sup.35 S, or .sup.125 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).

Anti-vertebrate fused antibodies also are useful for the affinity purification of vertebrate fused from recombinant cell culture or natural sources. In this process, the antibodies against vertebrate fused are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the vertebrate fused to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the vertebrate fused, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the vertebrate fused from the antibody.

H. Fused Antagonists

Several approaches may be suitably employed to create the fused antagonist and agonist compounds of the present invention. Any approach where the antagonist molecule can be targeted to the interior of the cell, which interferes or prevents wild type fused from normal operation is suitable. For example, competitive inhibitors, including mutant fused such as dominant negative mutant identified in the Examples, which prevent fused from properly binding with other proteins necessary for Hh signaling. Additional properties of such antagonist or agonist molecules are readily determinable by one of ordinary skill, such as size, charge and hydrophobicity suitable for transmembrane transport.

Where mimics or other mammalian homologues of fused are to be identified or evaluated, the cells are exposed to the test compound and compared to positive controls which are exposed only to human fused, and to negative controls which were not exposed to either the compound or the natural ligand. Where antagonists or agonists of fused signal modulation are to be identified or evaluated, the cells are exposed to the compound of the invention in the presence of the natural ligand and compared to controls which are not exposed to the test compound.

Detection assays may by employed as a primary screen to evaluate the phosphatase inhibition/enhancing activity of the antagonist/agonist compounds of the invention. The assays may also be used to assess the relative potency of a compound by testing a range of concentrations, in a range from 100 mM to 1 pM, for example, and computing the concentration at which the amount of phosphorylation or signal transduction is reduced or increased by 50% (IC.sub.50) compared to controls.

Assays can be performed to identify compounds that affect phosphorylation of fused substrates. Specifically, assays can be performed to identify compounds that increase the phosphorylation activity of fused or assays can be performed to identify compounds that decrease the phosphorylation of fused substrates. These assays can be performed either on whole cells themselves or on cell extracts. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, cell based assays, etc. Such assay formats are well known in the art.

The screening assays of the present invention are amenable to high-throughput screening of chemical libraries, and are particularly suitable for identifying small molecule drug candidates.

(1) Antagonist and agonist molecules

To screen for antagonists and/or agonists of fused signaling, the assay mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, fused induces hedgehog signaling with a reference activity. The mixture components can be added in any order that provides for the requisite hedgehog activity. Incubation may be performed at any temperature that facilitates optimal binding, typically between about 4.degree. and 40.degree. C., more commonly between about 15.degree. and 40.degree. C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening, and are typically between about 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours. After incubation, the effect of the candidate pharmacological agent on the fused signaling is determined in any convenient way. For cell-free binding-type assays, a separation step is often used to separate bound and unbound components. Separation may, for example, be effected by precipitation (e.g. TCA precipitation, immunoprecipitation, etc.), immobilization (e.g. on a solid substrate), followed by washing. The bound protein is conveniently detected by taking advantage of a detectable label attached to it, e.g. by measuring radioactive emission, optical or electron density, or by indirect detection using, eg. antibody conjugates.

For example, a method of screening for suitable fused antagonists and/or agonists could involve the application of agents present in the fused activating Gli reporter assay described in the Examples. Such a screening assay could compare in situ hybridization in the presence and absence of the candidate antagonist and/or agonist in a fused expressing tissue as well as confirmation or absence of fused modulated cellular development. Typically these methods involve exposing an immobilized fused to a molecule suspected of binding thereto and determining binding or phosphorylation of the molecule to the immobilized fused and/or evaluating whether or not the molecule activates (or blocks activation of) fused. In order to identify such fused binding ligands, fused can be expressed on the surface of a cell and used to screen libraries of synthetic candidate compounds or naturally-occurring compounds (e.g., from endogenous sources such as serum or cells).

Suitable molecules that affect the protein-protein interaction of fused and its binding proteins include fragments of the latter or small molecules, e.g., peptidomimetics, which will prevent interaction and proper complex formation. Such small molecules, which are usually less than 10K molecular weight, are preferable as therapeutics since they are more likely to be permeable to cells, are less susceptible to degradation by various cellular mechanisms, and are not as apt to elicit an immune response as proteins. Small molecules include but are not limited to synthetic organic or inorganic compounds. Many pharmaceutical companies have extensive libraries of such molecules, which can be conveniently screened by using the assays of the present invention. Non-limiting examples include proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosacchardies, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like.

A preferred technique for identifying molecules which bind to fused utilizes a chimeric substrate (e.g., epitope-tagged fused or fused immunoadhesin) attached to a solid phase, such as the well of an assay plate. The binding of the candidate molecules, which are optionally labeled (e.g., radiolabeled), to the immobilized receptor can be measured. Alternatively, competition for activation of Gli can be measured. In screening for antagonists and/or agonists, fused can be exposed to a fused substrate followed by the putative antagonist and/or agonist, or the fused binding protein and antagonist and/or agonist can be added simultaneously, and the ability of the antagonist and/or agonist to block fused activation can be evaluated.

(2) Detection assays

The fused polypeptides are useful in assays for identifying lead compounds for therapeutically active agents that modulate fused hedgehog signaling. Specifically, lead compounds that either prevent the formation of fused signaling complexes or prevent or attenuate fused modulated hedgehog signaling (e.g, binding to fused itself or to a substrate) can be conveniently identified.

Various procedures known in the art may be used for identifying, evaluating or assaying the inhibition of activity of the fused proteins of the invention. As fused is believed to operate in a similar manner as other kinases, techniques known for use with identifying kinase/phosphatase modulators may also be employed with the present invention. In general, such assays involve exposing target cells in culture to the compounds and a) biochemically analyzing cell lysates to assess the level and/or identity of phosphorylation; or (b) scoring phenotypic or functional changes in treated cells as compared to control cells that were not exposed to the test substance. Such screening assays are described in U.S. Pat. No. 5,602171, U.S. Pat. No. 5,710,173, WO 96/35124 and WO 96/40276.

(a) Biochemical detection techniques

Biochemical analysis techniques can be evaluated by a variety of techniques. One typical assay mixture which can be used with the present invention contains fused and a protein with which fused is normally associated (e.g. Gli), usually in an isolated, partially pure or pure form. One or both of these components may be fused to another peptide or polypeptide, which may, for example, provide or enhance protein--protein binding, improve stability under assay conditions, etc. In addition, one of the components usually comprises or is coupled to a detectable label. The label may provide for direct detection by measuring radioactivity, luminescence, optical or electron density, etc., or indirect detection such as an epitope tag, an enzyme, etc. The assay mixture can additionally comprise a candidate pharmacological agent, and optionally a variety of other components, such as salts, buffers, carrier proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., which facilitate binding, increase stability, reduce non-specific or background interactions, or otherwise improve the efficiency or sensitivity of the assay.

The following detection methods may also be used in a cell-free system wherein cell lysate containing the signal transducing substrate molecule and fused is mixed with a compound of the invention. The substrate is phosphorylated by initiating the kinase reaction by the addition of adenosine triphosphate (ATP). To assess the activity of the compound, the reaction mixture may be analyzed by the SDS-PAGE technique or it may be added to substrate-specific anchoring antibody bound to a solid support, and a detection procedure as described above is performed on the separated or captured substrate to assess the presence or absence of pSer/Thr. The results are compared to those obtained with reaction mixtures to which the compound is not added. The cell-free system does not required the natural ligand or knowledge of its identity. The cell-free system does not require mixtures to which the compound is not added. The cell-free system does not require the natural ligand or knowledge of its identity. For example, Posner et al. (U.S. Pat. No. 5,155,031 describes the use of insulin receptor as a substrate and rat adipocytes as target cells to demonstrate the ability of pervanadate to inhibit PTP activity. Another example, Burke et al., Biochem. Biophys. Res. Comm. 204:129-134 (1994) describes the use of autophosphorylated insulin receptor and recombinant PTP1B in assessing the inhibitory activity of a phophotyrosyl mimetic.

(i) Whole cell detection

A common technique involves incubating cells with vertebrate fused and radiolabeled phosphate, lysing the cells, separating cellular protein components of the lysate using an SDS-polyacrylamide gel (SDS-PAGE) technique, in either one or two dimensions, and detecting the presence of phosphorylated proteins by exposing X-ray film. Detection can also be effected without using radioactive labeling. In such a technique, the protein components (e.g., separated by SDS-PAGE) are transferred to a nitrocellulose membrane where the presence of phosphorylated serine/threonines is detected using an antiphosphoserine/threonine antibody (anti-pS/T).

Alternatively, the anti-pS/T can be conjugated with an enzyme, such as horseradish peroxidase, and detected by subsequent addition of a colorimetric substrate for the enzyme. A further alternative involves detecting the anti-PS/T by reacting with a second antibody that recognizes the anti-PS/T, this second antibody being labeled with either a radioactive moiety or an enzyme as previously described. Examples of these and similar techniques are described in Hansen et al., Electrophoresis 14: 112-126 (1993); Campbell et al., J. Biol. Chem. 268: 7427-7434 (1993); Donato et al., Cell Growth Diff. 3: 258-268 (1992); Katagiri et al., J. Immunol. 150: 585-593 (1993). Additionally, the anti-pS/T can be detected by labeling it with a radioactive substance, followed by scanning the labeled nitrocellulose to detect radioactivity or exposure of X-ray film.

(ii) Kinase assays

When the screening methods of the present invention for fused antagonists/agonists are carried out as an ex vivo assay, the target kinase (e.g. fused) can be a substantially purified polypeptide. The kinase substrate (e.g., MBP, Gli) is a substantially purified substrate, which in the assay is phosphorylated in a reaction with a substantially purified phosphate source that is catalyzed by the kinase. The extent of phosphorylation is determined by measuring the amount of substrate phosphorylated in the reaction. A variety of possible substrates may be used, including the kinase itself in which instance the phosphorylation reaction measured in the assay is autophosphorylation. Exogenous substrates may also be used, including standard protein substrates such as myelin basic protein (MBP); yeast protein substrates; synthetic peptide substrates, and polymer substrates. Of these, MBP and other standard protein substrates may be regarded as preferred (see Example 10). Other substrates may be identified, however, which are superior by way of affinity for the kinase, minimal perturbation of reaction kinetics, possession of single or homogenous reaction sites, ease of handling and post-reaction recover, potential for strong signal generation, and resistance or inertness to test compounds.

Measurement of the amount of substrate phosphorylated in the ex vivo assay of the invention may be carried out by means of immunoassay, radioassay or other well-known methods. In an immunoassay measurement, an antibody (such as a goat or mouse anti-phosphoserine/threonine antibody) may be used which is specific for phosphorylated moieties formed during the reaction. Using well-known ELISA techniques, the phosphoserine/threonine antibody complex would itself be detected by a further antibody linked to a label capable of developing a measurable signal (as for example a fluorescent or radioactive label). Additionally, ELISA-type assays in microtitre plates may be used to test purified substrates. Peraldi et al, J. Biochem. 285: 71-78 (1992); Schraag et al., Anal. Biochem. 211: 233-239 (1993); Cleavland, Anal. Biochem. 190: 249-253 (1990); Farley, Anal. Biochem. 203: 151-157 (1992) and Lozaro, Anal. Biochem. 192: 257-261 (1991).

For example, detection schemes can measure substrate depletion during the kinase reaction. Initially, the phosphate source may be radiolabeled with an isotope such as .sup.32 P or .sup.33 P, and the amount of substrate phosphorylation may be measured by determining the amount of radiolabel incorporated into the substrate during the reaction. Detection may be accomplished by: (a) commercially available scintillant-containing plates and beads using a beta-counter, after adsorption to a filter or a microtitre well surface, or (b) photometric means after binding to a scintillation proximity assay bead or scintillant plate. Weernink and Kiijken, J. Biochem. Biophs. Methods 31: 49, 1996; Braunwalder et al., Anal. Biochem. 234: 23 (1996); Kentrup et al., J. Biol. Chem. 271: 3488 (1996) and Rusken et al., Meth. Enzymol. 200: 98 (1991).

Preferably, the substrate is attached to a solid support surface by means of non-specific or, preferably, specific binding. Such attachment permits separation of the phosphorylated substrate from unincorporated, labeled phosphate source (such as adenosine triphosphate prior to signal detection. In one embodiment, the substrate may be physically immobilized prior to reaction, as through the use of Nunc.TM. high protein binding plate (Hanke et al., J. Biol. Chem. 271: 695 (1996)) or Wallac ScintiStrip.TM. plates (Braunwalder et al., Anal. Biochem. 234: 23 (1996). Substrate may also be immobilized after reaction by capture on, for example, P81 phophocellulose (for basic peptides), PEI/acidic molybdate resin or DEAE, or TCA precipitation onto Whatman.TM. 3MM paper, Tiganis et al., Arch. Biochem. Biophys. 325: 289 (1996); Morawetz et al., Mol. Gen. Genet. 250; 17 (1996); Budde et al, Int J. Pharmacognosy 33: 27 (1995) and Casnellie, Meth. Enz. 200: 115 (1991). Yet another possibility is the attachment of the substrate to the support surface, as by conjugation with binding partners such as glutathione and streptavidin (in the case of GST and biotin), respectively) which have been attached to the support, or via antibodies specific for the tags which are likewise attached to the support.

Further detection methods may be developed which are preferred to those described above. Especially for use in connection with high-throughput screening, it is expected that such methods would exhibit good sensitivity and specificity, extended linear range, low background signal, minimal fluctuation, compatibility with other reagents, and compatibility with automated handling systems.

The in vivo efficacy of the treatment of the present invention can be studied against chemically induced tumors in various rodent models. Tumor cell lines propagated in in vitro cell cultures can be introduced in experimental rodents, e.g. mice by injection, for example by the subcutaneous route. Techniques for chemical inducement of tumors in experimental animals are well known in the art.

(b) Biological detection techniques:

The ability of the antagonist/agonist compounds of the invention to modulate the activity fused, which itself modulates hedgehog signaling, may also be measured by scoring for morphological or functional changes associated with ligand binding. Any qualitative or quantitative technique known in the art may be applied for observing and measuring cellular processes which comes under the control of fused. The activity of the compounds of the invention can also be assessed in animals using experimental models of disorders caused by or related to dysfunctional hedgehog signaling. For example, ineffective DHh hedgehog signaling in mice leads to viable but sterile mice. The effects of mutant fused (hfused-DN) also affects gut development, which is regulated by IHh expression. Additionally, proper SHh signaling is critical to murine embryonic development at the notochord and floor plate, neural tube, distal limb structures, spinal column and ribs. Improper SHh signaling, is also correlative with cyclopia. Any of these phenotypic properties could be evaluated and quantified in a screening assay for fused antagonists and/or agonist. Disease states associated with overexpression of hedgehog is associated with basal cell carcinoma while inactive sonic hedgehog signaling leads to improper neural development.

The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the compounds of the invention should lie within a range of circulating concentrations with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration.

(2) Antisense nucleotides

Another preferred class of antagonists involves the use of gene therapy techniques, include the administration of antisense nucleotides. Applicable gene therapy techniques include single or multiple administrations of therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. Short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by restricted uptake by the cell membrane, Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 (1986). The oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups by uncharged groups.

There are a variety of techniques known for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, ex vivo, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection, Dzau et al., Trends Biotech. 11: 205-210 (1993). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432 (1987); Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990). For a review of known gene marking and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992).

In one embodiment, fused antagonist and/or agonist molecules may be used to bind endogenous ligand in the cell, thereby causing the cell to be unresponsive to fused wild type, especially when the levels of fused in the cell exceed normal physiological levels. Also, it may be beneficial to bind endogenous fused substrates or complexing agents that are activating undesired cellular responses (such as proliferation of tumor cells).

In a further embodiment of the invention, fused expression may be reduced by providing fused-expressing cells with an amount of fused antisense RNA or DNA effective to reduce expression of the fused protein.

I. Diagnostic Uses

Another use of the compounds of the invention (e.g., human and vertebrate fused, vertebrate fused variant and anti-vertebrate fused antibodies) described herein is to help diagnose whether a disorder is driven, to some extent, fused or hedgehog signaling. For example, basal cell carcinoma cells are associated with active hedgehog signaling.

A diagnostic assay to determine whether a particular disorder is driven by hedgehog signaling, can be carried out using the following steps: (1) culturing test cells or tissues; (2) administering a compound which can inhibit fused modulated hedgehog signaling; and (3) measuring the degree of kinase attenuation on the fused substrate in cell lysates or hedgehog mediated phenotypic effects in the test cells. The steps can be carried out using standard techniques in light of the present disclosure. For example, standard techniques can be used to isolate cells or tissues and culturing or in vivo.

Compounds of varying degree of selectivity are useful for diagnosing the role of fused. For example, compounds which inhibit fused in addition to another form of kinase can be used as an initial test compound to determine if one of several serine/threonine kinases drive the disorder. The selective compounds can then be used to further eliminate the possible role of the other serine/threonine kinases in driving the disorder. Test compounds should be more potent in inhibiting serine/threonine kinase activity than in exerting a cytotoxic effect (e.g., an IC.sub.50 /LD.sub.50 of greater than one). The IC.sub.50 and LD.sub.50 can be measured by standard techniques, such as an MTT assay, or by measuring the amount of LDH released. The degree of IC.sub.50 /LD.sub.50 of a compound should be taken into account in evaluating the diagnostic assay. Generally, the larger the ratio the more relative the information. Appropriate controls take into account the possible cytotoxic effect of a compound of a compound, such as treating cells not associated with a cell proliferative disorder (e.g., control cells) with a test compound, can also be used as part of the diagnostic assay. The diagnostic methods of the invention involve the screening for agents that modulate the effects of fused upon hedgehog signaling. Exemplary detection techniques include radioactive labeling and immunoprecipitating (U.S. Pat. No. 5,385,915).

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Rockville, Md.

EXAMPLE 1

Isolation of human fused cDNA clones

An expressed sequence tag (EST) DNA database (LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.) was searched for a human homologue of the Drosophila segment polarity gene fused (SEQ ID NO:26) (Preat et al., Nature 347: 87-9 (1990)). The EST Incyte #2515662 (FIG. 2 (SEQ ID NO:3)) was identified as a potential candidate. In order to identify human cDNA libraries containing human fused clones, human cDNA libraries in pRK5 were first screened by PCR using the following primers:

h-FUSED.f (SEQ ID NO:8) 5'-CAATACAATGGTGCTGACATCCATCAAAGGCA-3'

h-FUSED.r (SEQ ID NO:9) 5'-'GAAGGGAGGGGTGCCTACTGCCA-3'

A fetal lung library was selected and enriched for fused cDNA clones by extension of single stranded DNA from plasmid libraries grown in dug.sup.- /bung.sup.- host using the h-FUSED.f primer in a reaction containing 10 .mu.l of 10.times. PCR Buffer (Perkin Elmer), 1 .mu.l dNTP (20 mM), 1 .mu.l library DNA (200 ng), 0.5 ml primer, 86.5 .mu.l H.sub.2 O and 1 .mu.l of Amplitaq.RTM. (Perkin Elmer) added after a hot start. The reaction was denatured for 1 min. at 95.degree. C., annealed for 1 min. at 60.degree. C. then extended for 20 min. at 72.degree. C. DNA was extracted with phenol/CHCl.sub.3, ethanol precipitated, then transformed by electroporation into DH10B host bacteria. Colonies from each transformation were plated and lifted on nylon membranes and screened with an oligo probe derived from the EST sequence of the following sequence:

h-FUSED.p (SEQ ID NO:10) 5'-CTCCAGCTCTGGAGACATATAGAGTGGTGTGCCTTTGA-3'

The oligo probe was labeled with [.gamma.-.sup.32 P]-ATP and T4 polynucleotide kinase. Filters were hybridized overnight at 42.degree. C. in 50% formamide, 5.times. SSC, 10.times. Denhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 .mu.g/ml of sonicated salmon sperm DNA. The filters were then rinsed in 2.times. SSC and washed in 0.1.times. SSC, 0.1% SDS then exposed to Kodak.RTM. X Ray films. Two positive clones (DNA28494 and DNA28495-FIGS. 4 & 5 (SEQ ID NOS:6 and 4), respectively) containing an insert of approximately 5 kb were isolated and sequenced. The sequence of clone DNA28495 (SEQ ID NO:4) contains a potential initiation methionine at position 116 followed by an open reading frame of 1944 bp (FIG. 4). However, this open reading frame (ORF) encodes a protein that is only 648 amino acids long, somewhat shorter than the 795 amino acid sequence of the Drosophila fused. Interestingly, a second open reading frame is present in the 3' region of the cDNA, from nucleotide 2295 to 4349 (FIG. 4), which suggests that the cDNA may have been improperly spliced and that an intron remains between the 2 ORFs, or correspond to an alternatively spliced variant of fused. The sequence of clone DNA28494 (SEQ ID NO:6) is very similar. There is one nucleotide difference between clone DNA28495 (SEQ ID NO:4) and clone DNA28494 (SEQ ID NO:6) located in the first ORF at position 1863 of clone 28495 (A vs. G) which changes the coding sequence from an Gln to a Arg at position 583. (FIG. 4). This change is likely due to an allelic variation. The first open reading frame of DNA28494 (SEQ ID NO:6) starts at residue 115 and is followed by a 647 amino acid long open reading frame. The sequences are identical except for the one change described above at position 583 and for the last 9 residues in the first open reading frame.

EXAMPLE 2

Expression of fused clones

In order to determine the size of the protein expressed from the cDNA corresponding to DNA28495 and DNA28494 (SEQ ID NOS:4 and 6), respectively, an HA epitope tag was inserted at the N-terminus of the protein by PCR using the following primers:

Hfus.Cla-HA.F: (SEQ ID NO: 11) 5'-CCATCGATGTACCCATACGACGTCCCAGACTACGCTGAAAAGTACCACGTGTTGGAGATG-3'

and hFus.Xba.R: (SEQ ID NO: 12) 5'-GCTCTAGACTAAGGGGCAGGTCCTGTGTTCTG-3'.

The PCR product was purified, digested with ClaI-SmaI and subcloned into the pRK5 plasmids containing DNA28494 and DNA28495 (SEQ ID NO:6 and 4), respectively . DNA from each of the constructs was transfected overnight into 293 cells using the CaPO4 method (Sambrook et al, supra; Ausuble et al., supra). After about 24 h. to 48 h. after transfection, the cells were harvested and the cell pellet was lysed in 1 ml of lysine buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, Aprotinin, Leupeptin, -PMSF, 1 mM NaF and 1 mM Sodium Vanadate) for 20 min at 4.degree. C. The extract was spun for 10 min at 10K then the supernatant was transferred to a new tube and precleared with 20 .mu.l Protein A sepharose for 1 h. The protein A sepharose was spun down and 1 .mu.l of anti-HA antibody (5 .mu.g, Boehringer) was added to each tube. After overnight incubation at 4.degree. C., 30 .mu.l of Protein G sepharose was added and the tubes incubated at 4.degree. C. for 1 hour. The protein G beads were then sun down for 1 min., washed 3 times with lysis buffer, resuspended in 20 .mu.l of laemli buffer in the presence of .beta.-mercapto ethanol. Samples were denatured for 5 min. at 100.degree. C. then loaded on a 6% polyacrylamide gel. Proteins were then transferred to nitrocellulose and analyzed by Western blot using the same anti-HA antibody overnight at 1 .mu.g/ml in blocking buffer (PBS, 0.5% Tween.RTM., 5% non fat dry milk, 3% goat serum followed by an anti-mouse HRP. ECL was used for the detection and the membrane was exposed for 90 seconds to X-Ray films. A specific band of 150 kDa was detected in the cell pellet of cells transfected with the construct with construct corresponding to clone DNA28494 (SEQ ID NO:6) and a specific band of approximately 100 kDa could be detected for clone DNA28495 (SEQ ID NO:4) (FIG. 6). These bands were not present in the mock transfected control. The presence of the 150 kDa band suggests the two open reading frames of DNA28494 (SEQ ID NO:6) can be spliced together to direct the synthesis of a large protein of 150 kDa. The absence of this band for DNA28495 (SEQ ID NO:4) suggested that this clone apparently cannot be correctly spliced. Alternative splicing of the fused gene seems to lead to the production of several different products and may be a mechanism or regulation of fused activity. Specific regions at the C-terminus of the Drosophila fused protein is known to be required for the activity of the molecule, Therond et al., Genetics 142: 1181-1198 (1996); Robbins et al., Cell 90: 225-234 (1997). Shorter fused molecules truncated at the C-terminus may therefore correspond to inactive or to dominant negative forms of the molecule.

EXAMPLE 3

Northern Blots

In order to determine the best tissue source to isolate more fused cDNAs and to identify a transcript encoding a full length 150 kDa fused molecule, human multiple tissue northern blots I, II and fetal blot from Clontech were probed with a 1.6 kb, ClaI-AccI fragment derived from clone DNA28494 (SEQ ID NO:6) labeled by random priming. The blots were hybridized in 50% formamide, 5.times. SSC, 10.times. Denhardt's, 0.05M Sodium phosphate (pH 6.5), 0.1% Sodium pyrophosphate, 50 mg/ml sonicated salmon sperm DNA, all in the presence of 1.times.10.sup.6 cpm/ml .sup.32 P-labeled probe at 42.degree. C. overnight. The blots were washed in 2.times. SSC at RT for 10 minutes and washed in 0.2.times. SSC/0.1% SDS at 42.degree. C. for 30 minutes then exposed to x-ray film overnight. FIG. 7 shows that the fused message is expressed at high levels in testis and at low levels in most other tissues, including fetal tissues. (FIG. 7).

EXAMPLE 4

PCR on different tissues to identify the correct splice form

In order to isolate a cDNA where the 2 potential ORFs were spliced together correctly, we designed the following primers flanking the potential intron and amplified various tissues including human fetal brain, brain, keratinocyte, testis, ovary, fetal liver, and lung templates.

F1 (SEQ ID NO:13) 5'-CTGACGACACAGCAGGTTGTC-3'

R4 (SEQ ID NO:14) 5'-CAGATGCTTCAGGATGGACAT-3'

Two microliters of each cDNA library was used as the template and PCR was done with Klentaq.RTM. polymerase. PCR was performed for 45 cycles of amplification with 94.degree. C. denaturation for 1 min., 55.degree. C. annealing for 1 min., and 68.degree. C. extensions for 2 min. One fifth of the reaction was loaded on 1% agarose gel and was Southern blotted. The blot was hybridized overnight with full-length fused probe labeled by random priming as described for the Northern blot.

A 1 kb PCR fragment was identified in fetal brain, testis and ovary. This fragment was gel-purified and subjected to direct PCR sequencing using both the F1 and R4 primer identified above as well a the following primers:

hf16 (SEQ ID NO:15) 5'-AGAGTAGCAACGTCACTGC-3'

hf8 (SEQ ID NO:16) 5'-CCTCACTGACAAGGCAGCAGG-3'

hf19 (SEQ ID NO: 17) 5'-CCCGAGGAGGCATCTGCACAG-3'

The sequence of this 1 kb fragment revealed that intron sequences were absent and that the 2 ORFs were connected together in the same reading frame. The sequence of the correctly spliced sequence is shown in FIG. 1 (SEQ ID NO:1). The initiator ATG is present at position 161 and is followed by an ORF of 3945 nucleotides which encodes a 1315 amino acid long protein with a predicted molecular weight of 144 kDa.

The overall similarity with Drosophila fused (SEQ ID NO:23) is 28% (FIG. 2). The N-terminal 263 amino acid domain of the protein containing the kinase domain is 55% homologous to the Drosophila fused kinase domain. The remaining 1052 amino acids portion of the protein is not appreciably homologous to other known proteins and, interestingly, is not homologous to the corresponding region in Drosophila fused. Interestingly, this region of non-homology includes the very C-terminus of the fly protein which appears to be required for activity, Robbins et al., Cell 90: 225-34 (1997); Therond et al., Genetics 142: 1181-98 (1996). The improperly spliced cDNAs described above may reflect alternative splicing of the fused gene which leads to the production of a molecule with a truncated C-terminus and may be a mechanism to regulate fused activity.

EXAMPLE 5

Reconstitution of the correctly spliced full length human fused

The fused clone DNA28495 was subcloned from the pRK5B plasmid into pRK5.tkneo using ClaI-HindIII. PCR was performed using human testis cDNA as a template and the primers hf3 (SEQ ID NO:18) (CAGAACTTCAGGTCCTAAAGG) and R4 (sequence see above, Example 4). PCR conditions were 45 cycles of (94.degree. C., 1 min, 46.degree. C. to 68.degree. C. temperature gradient annealing for 1 min, and 68.degree. C., 4 min). The PCR fragment was digested with AccI and ligated in the pRK5.tkneo.fused plasmid cut with AccI in order to replace the region containing the intron with the correct spliced form. Two subclones were sequenced between the two AccI site and had the same correct sequence.

EXAMPLE 6

In situ hybridization

E11.3 and E13.5 mouse embryos were immersion-fixed overnight at 4.degree. C. in 4% paraformaldehyde, cryoprotected overnight in 15% sucrose, embedded in O.T.C. and frozen on liquid nitrogen. Adult mouse brains were fresh frozen with powdered dry ice. P1 mouse brains, adult mouse testis and adult rat spinal cords were embedded in O.T.C. and frozen on liquid nitrogen. Sections were cut at 16 mm, and processed for in situ hybridization for fused by the method of Phillips et al.. Science 250: 290-294 (1990). RNA probes were labeled with .sup.33 P-UTP as described by Melton et al., Nucleic Acids Res. 12: 7035-7052 (1984). Sense and antisense probes were synthesized from a mouse fused DNA fragment using T3 and T7, respectively, corresponding to the region encoding amino acid residues 317-486 of the human sequence.

FIG. 8 reveals that the mouse fused mRNA is widely distributed in SHh responsive tissues, including the neural tube, pre-somitic mesoderm, somites, developing limb buds and skin. Transcripts for fused were also found in the embryonic gut, testis, cartilage and muscle--Tissues that are exposed to the other members of the Hh protein family; Desert and Indian. In the E11-5 mouse nervous system, high levels of fused transcripts were detected throughout the forebrain, midbrain, hindbrain and spinal cord. These high levels of expression were retained in embryonic day 13.5. In both embryonic days 11.5 and 13.5, fused mRNA was detected mainly in the ventral aspect of the neural tube, in regions that are likely to be exposed to the ventral midline-derived SHh. By post natal day -1, widespread expression of fused is still maintained throughout the brain with high levels of transcripts detected in the cortex, hypocampus, ependima and choroid plexus. In the adult, low levels of fused expression are detected all through the brain with higher levels confined to the ependima.

The tissue distribution of fused and the Hh receptor components, Smo and Ptch show considerable overlap. All of them are initially expressed through the neural tube as well as in other Hh responsive tissues. However, whereas Smo mRNA was evenly distributed along the dorso-ventral axis, Ptch and fused mRNAs are found at higher levels ventrally, suggesting that they may be upregulated by Hh. In addition while by day E12, expression of both Smo and Ptch is found mainly in cells which are in close proximity to the ventricular zone, fused mRNA is still widely expressed and its levels decline only later. In the adult expression of both Smo and fused is confined to the ependima where neurogenesis continues.

Detailed analysis of fused expression in adult testis was also performed by in situ hybridization (FIG. 9). Fused was found to be expressed at very high levels on stages I and II germ cells in the seminiferous tubules. Levels of fused vary in different seminiferous tubules, suggesting that its expression is regulated according to the germinal cell state of differentiation.

EXAMPLE 7

Gli Luciferase Assay

Given the low homology between dfused and hfused, it was prudent to determine whether in fact the isolated hfused is indeed a mediator of Hh signaling. The following assay was developed to measure the activation of the transcription factor GLI, the mammalian homologue of the Drosophila cubitus interruptus (Ci). It has been shown that GLI is a transcription factor activated upon SHh stimulation of cells.

Nine (9) copies of a GLI binding site present in the HNF3.beta. enhancer, (Sasaki et al., Development 124: 1313-1322 (1997)), were introduced in front of a thymidine kinase minimal promoter driving the luciferase reporter gene in the pGL3 plasmid (Promega). The sequence of the GLI binding sequence was: TCGACAAGCAGGGAACACCCAAGTAGAAGCTC (p9XGliLuc) (SEQ ID NO:19), while the negative control sequence was: TCGACAAGCAGGGAAGTGGGAAGTAGAAGCTC (p9XmGliLuc) (SEQ ID NO:20). These constructs were cotransfected with the full length fused construct or with a plasmid encoding sonic hedgehog in C3H10T1/2 cells grown in F12, DMEM (50:50), 10% FCS heat inactivated. The day before transfection 1.times.10.sup.5 cells per well was inoculated in 6 well plates, in 2 ml of media. The following day, 1 .mu.g of each construct was cotransfected in duplicate with 0.025 mg ptkRenilla luciferase plasmid using lipofectamine (Gibco-BRL) in 100 .mu.l OptiMem (with GlutaMAX) as per manufacturer's instructions for 3 hours at 37.degree. C. Serum (20%, 1 ml) was then added to each well and the cells were incubated for 3 more hours at 37.degree. C. Cells were then washed twice with PBS, then incubated for 48 hours at 37.degree. C. in 2 ml of media. Each well was then washed with PBS, and the cells lysed in 0.5 ml Passive Lysis Buffer (Promega) for 15 min. at room temperature on a shaker. The lysate was transferred in eppendorf tubes on ice, spun in a refrigerated centrifuge for 30 seconds and the supernatant saved on ice. For each measure, 20 .mu.l of cell lysate was added to 100 .mu.l of LARII (luciferase assay reagent, Promega) in a polypropylene tube and the luciferase light activity measured. The reaction was stopped by the addition of Stop and Glow buffer (Promega), mixed by pipetting up and down 3 to 5 times and Renilla luciferase lights activity was measured on the luminometer.

As shown in FIG. 6, fused can induce GLI activity (9.5 fold) in a similar manner as SHh (5.5 fold) This result suggests that the fused gene isolated is a mediator of SHh signaling. An irrelevant serine-threonine kinase, Akt, was not active in this assay (data not shown). The fused activity is dependent on an intact kinase domain as molecules with deletion of this region (fused C-term (SEQ ID NO:27)) or mutation of a conserved lysine residue at about amino acid position 33 in the ATP binding site (fused-DN (SEQ ID NO:25)) were not able to activate GLI. Similarly, the C-terminal tail of the protein is necessary for this activity since the kinase domain alone was not active in this assay (fused KD (SEQ ID NO:24)). Expression of each protein was verified by Western blot using an HA tag inserted at the N-terminus of the molecule (data not shown). These results substantiate the conclusion that the homologue of the dfused isolated by Applicants is indeed hfused. Furthermore, these results indicate that fused is capable of and sufficient for the activation of Gli, the major target of SHh signaling and is thus likely to be a direct mediator of the SHh signal in vertebrates.

EXAMPLE 8

Induced cyclopia in frog embryos

Introduction:

In order to demonstrate that the human fused gene is not only capable of but also required to transduce the SHh signal in vertebrates, a mutant version of fused known as fused-DN (dominant negative) having a mutation of the lysine at position 33 in the ATP binding site was created (SEQ ID NO:25). This residue is conserved among all kinases and is necessary for kinase activity (Hanks et al., Methods Enzymol. 200: 38-62 (1991) and its conversion to any other residue in most cases results in the creation of dominant negative mutants.

Methods:

Plasmid Construction:

Wild type fused cDNA with an HA tag inserted at the carboxy terminus was subcloned into pRK5 and a dominant negative form was generated by conversion of lysine at positive 33 to an arginine. Supercoiled plasmid DNA was prepared by Qiagen and used for injection into Xenopus laevis embryo.

Manipulation of Xenopus embryos:

Adult female frogs were boosted with 200 I.U. pregnant mare serum 3 days before use and with 800 I.U. of human chorionic gonadotropin the night before injection. Fresh oocytes were squeezed out from female frogs the next morning and in vitro fertilization of oocytes was performed by mixing oocytes with minced testis from sacrificed male frogs. Developing embryos were maintained and staged according to Nieuwkoop and Faber, Normal Table of Xenopus laevis, N.-H. P. Co., ed. (Amsterdam, 1967).

Fertilized eggs were dejellied with 2% cysteine (pH 7.8) for 10 minutes, washed once with distilled water and transferred to 0.1.times. MBS with 5% Ficoll. Fertilized eggs were lined on injection trays in 0.1.times. MBS with 5% Ficoll. Two-cell stage developing Xenopus embryos were injected with 200 pg of either pRK5 containing wild type fused (WT) (SEQ ID NO:1) or dominant negative fused (DN) (SEQ ID NO:25). Injected embryos were kept on trays for another 6 hours, after which they were transferred to 0.1.times. MBS with 50 mg/ml gentamycin for 3 days until reaching Nieukwkoop stage 35 when eye development is complete.

Results:

To test whether human fused gene acts as a signal transducer of Hedgehog signaling, we injected wild type (SEQ ID NO:2) or dominant negative form (SEQ ID NO:25) of human fused in developing frog embryos. Embryos injected with 120 pg of DNA divided normally in blastula stage and gastrulate normally. While eye development was normal in wild type, fused (SEQ ID NO:2) injected and mock injected embryos, about 30% (Table 1) of the embryos that were injected with fused-DN showed fused eye structure or two eyes connected by some pigmented retina tissue (FIG. 11A). In Table 1, 200 pg of plasmid DNA was delivered to the animal pole of 2-cell stage embryos. Each sample represents the results of at least 3 independent experiments. Embryos were scored visually for cyclopia defects. 

                  TABLE 1
    ______________________________________
    Fusion-DN Induced Cyclopia in Xenopus Embryos
        Injected DNA      Normal    Cyclop
                                          n
    ______________________________________
    Hu-fused (SEQ ID NO:2)
                      45        0       45
      Kinase domain (SEQ ID NO:24) 43 0 43
      C-terminus (SEQ ID NO:27) 53 1 54
      fused DN (SEQ ID NO:25) 32 15   47
      Uninjected 61 0 61
    ______________________________________


The observed cyclopia phenotype is strikingly similar to the one of mouse embryos deficient in SHh (Chiang et al., Nature 383: 407-13 (1996) and of zebrafish embryos where SHh signaling has been blocked by overexpression of a constitutive active PKA, Hammerschmidt et al., Genes Dev. 10: 647-58 (1996); Ungar and Moon, Dev. Biol. 178: 186-91 (1996). In addition, both brain (forebrain) and gut development appeared normal at later stages of tadpole development in the fused-DN (SEQ ID NO:25) injected embryos (FIG. 11B). In contrast, embryos overexpressing either wild type fused (SEQ ID NO:2) or N or C-terminal terminal truncation mutants (SEQ ID NOS:27 and 24), respectively, did not present any abnormalities.

During normal development of the Xenopus eye, the eye primordium starts as a single field expressing transcription factor Pax-6, which is a vertebrate homologue of Drosophila eyeless, Li et al., Development. 124: 603-15 (1997). At the neurula stage, this eye field is separated into two eye primordia due to an inhibiting signal from prechordal mesoderm. It has been further demonstrated that SHh is the prechordal mesoderm derived signal that is responsible for the inhibition of Pax-6 expression in the midline of the eyefield.

To further understand how overexpression of fused-DN (SEQ ID NO:25) induced a fused eye in Xenopus embryos, whole mount in situ hybridization was performed in order to determine the expression pattern of Pax-6 in injected embryos. As shown in FIG. 11C, Pax-6 expression in embryos injected with fused-DN (SEQ ID NO:25) remains as a single field (FIG. 11D). Thus fused-DN (SEQ ID NO:25) induces a cyclopia phenotype by most likely preventing SHh from inhibiting Pax-6 expression in the midline of the eyefield.

EXAMPLE 9

Rescue of fused-DN Injected Xenopus Embryos by Gli

SHh expression in early floor plate cells is induced by SHh produced by the notochord. To test whether SHh expression in the floor plate will also be inhibited when SHh signaling is blocked, early neurula stage embryos injected with fused-DN or wild-type constructs were stained for SHh expression (See Example 8 for procedure). SHh expression in floor plate cells or early neurula stage embryos was completely suppressed in 26 out of 28 embryos injected when the mutated fused is overexpressed (Table 2, FIG. 11C, left embryo), while the expression of SHh was unaffected in control embryos (FIG. 6E, right embryo). Table 2 represents scored data from three independent experiments. 100 pg of fused-DN, 100 pg of fused-wt or 50 pg of Gli-1 plasmid were injected in 2-cell stage embryos. Embryos were harvested at early neurula stage for SHh staining. 

                  TABLE 2
    ______________________________________
    Wild type fused and Gli rescue SHh expression
      in floor plate when coexpressed with fused-DN
                      SHh staining
                                Percentage
    ______________________________________
    fused-DN (SEQ ID NO:25)
                       2/28     7%
      fused-DN + fused WT 20/24 83%
      fused-DN + Gli 36/36 100
    ______________________________________


To confirm that this phenotype was due to specific inhibition of the SHh signaling pathway in the floor plate, we attempted to rescue the phenotype by coinjection of wt fused RNA with fused-DN RNA in a 1:1 ratio. Table 2 shows that more than 80% of the embryos coinjected with wt fused and fused-DN RNAs show normal SHh staining in the floor plate. This demonstrates that SHh expression in fused-DN injected embryos is specifically blocked by inhibition of endogenous fused activity.

To further demonstrate that the observed phenotype of fused-DN are due to disruption of the SHh signal cascade and to confirm that hfused works upstream of Gli in this pathway, we asked whether the overexpression of Gli can also rescue the phenotype of Xenopus embryos injected with fused-DN. As shown in Table 2, the rescue of SHh expression in the floor plate of fused-DN injected embryos is complete when Gli is overexpressed. Taken together, these findings are consistent with Applicants hypothesis that vertebrate fused functions in the SHh pathway and that is a necessary mediator in the SHh signal transduction pathway, which acts upstream of Gli.

EXAMPLE 10

Immunoprecipitations and In Vitro Kinase Assay

To directly determine whether hfused has kinase activity, fused, fused-DN and fused-kd cDNAs were tagged with the influenza HA epitope tag and transiently transfected into 293 cells. Immunoprecipitates were tested for kinase activity in the presence of myelin basic protein (MBP) and [.gamma.-.sup.32 P]-ATP. The amount of 32P incorporated into MBP was determined after SDS-PAGE and found to be was about 3 times higher than in fused-KD (SEQ ID NO:25) and 2 times higher in wt fused (SEQ ID NO:2) containing extracts compared to controls, while mutation of Lys33 to Arg (fused-DN) (SEQ ID NO:25) neutralizes the activity (FIG. 12).

For immunoprecipitation experiments human embryonic kidney 293 cells were transiently transfected with the various expression plasmids. After 24 hours, the transfected cells were collected and lysed for 20 min. at 4.degree. C. in 1 ml of lysis buffer (50 mM Tris, pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF and protease inhibitors (Complete, Boehringer Mannheim) containing 1% NP-40, 0.5% deoxycholic acid. Cell debris was removed by centrifugation for 10 min. at 10,000 rpm and the sodium chloride concentration of the cell lysates was increased to 250 mM. The supernatant was precleared for 1 hour with 20 .mu.l Protein A Sepharose (Pharmacia). Lysates were immunoprecipitated using anti-HA antibodies followed by Protein A Sepharose. The beads were washed twice with lysis buffer containing 250 mM sodium chloride, twice with lysis buffer containing 1M sodium chloride, and then twice with kinase assay buffer (20 mM HEPES, pH 7.6), 1 mM DTT, 1 mM NaF and 1 mM sodium orthovanadate). After the last wash, the beads were resuspended in 20 .mu.l kinase assay buffer supplemented with 10 mCi [.gamma.-.sup.32 P]-ATP, 20 mM .beta.-glycerophosphate, 20 mM PNPP, 20 mM MgCl.sub.2, 1 mM EGTA, 100 .mu.M cold ATP and 0.5 mg/ml Myelin Basic Protein (Sigma), and incubated for 20 min. at 37.degree. C. Reactions were stopped with 20 .mu.l SDS-sample buffer, run on a denaturing 4-20% SDS polyacrylamide gel, and analyzed by phosphoimager.

EXAMPLE 11

Expression of fused in E. coli

The DNA sequence encoding human fused is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites that correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences that encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the vertebrate fused coding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.

After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized vertebrate fused protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.

EXAMPLE 12

Expression of fused in mammalian cells

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as the expression vector. Optionally, the vertebrate fused DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the vertebrate fused DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-fused.

In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 .mu.g pRK5-fused DNA is mixed with about 1 .mu.g DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227M CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed to form for 10 minutes at 25.degree. C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37.degree. C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 .mu.Ci/ml .sup.35 S-cysteine and 200 .mu.Ci/ml .sup.35 S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of vertebrate fused polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.

In an alternative technique, vertebrate fused may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 .mu.g pRK5-fused DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 .mu.g/ml bovine insulin and 0.1 .mu.g/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed vertebrate fused can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

In another embodiment, vertebrate fused can be expressed in CHO cells. The pSUi-fused can be transfected into CHO cells using known reagents such as CaPO.sub.4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as .sup.35 S-methionine. After determining the presence of vertebrate fused polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed vertebrate fused can then be concentrated and purified by any selected method.

Epitope-tagged vertebrate fused may also be expressed in host CHO cells. The vertebrate fused may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into an expression vector. The poly-his tagged vertebrate fused insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged vertebrate fused can then be concentrated and purified by any selected method, such as by Ni.sup.2+ -chelate affinity chromatography.

EXAMPLE 13

Expression of vertebrate fused in Yeast

The following method describes recombinant expression of vertebrate fused in yeast.

First, yeast expression vectors are constructed for intracellular production or secretion of vertebrate fused from the ADH2/GAPDH promoter. DNA encoding vertebrate fused, a selected signal peptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of vertebrate fused. For secretion, DNA encoding vertebrate fused can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leader sequence, and linker sequences (if needed) for expression of vertebrate fused.

Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant vertebrate fused can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing vertebrate fused may further be purified using selected column chromatography resins.

EXAMPLE 14

Expression of vertebrate fused in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of vertebrate fused in Baculovirus-infected insect cells.

The vertebrate fused is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the vertebrate fused or the desired portion of the vertebrate fused (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28.degree. C., the released viruses are harvested and used for further amplifications. Viral infection and protein expression is performed as described by O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).

Expressed poly-his tagged vertebrate fused can then be purified, for example, by Ni.sup.2+ -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl.sub.2 ; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A Ni.sup.2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A.sub.280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A.sub.280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or western blot with Ni.sup.2+ -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His.sub.10 -tagged vertebrate fused are pooled and dialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) vertebrate fused can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.

EXAMPLE 15

Preparation of Antibodies that Bind Vertebrate fused

This example illustrates preparation of monoclonal antibodies, which can specifically bind vertebrate fused.

Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified vertebrate fused, fusion proteins containing vertebrate fused, and cells expressing recombinant vertebrate fused on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the vertebrate fused immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect vertebrate fused antibodies.

After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of vertebrate fused. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity against vertebrate fused. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against vertebrate fused is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti- vertebrate fused monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

Deposit of Material

The following materials have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., USA (ATCC): 

    ______________________________________
    Designation     ATCC Dep. No.
                                Deposit Date
    ______________________________________
    pRK5tkneo.hFused-1272
                    209637      2/19/98
    ______________________________________


This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC 122 and the Commissioner's rules pursuant thereto (including 37 CFR 1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. 

    __________________________________________________________________________
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       - -       cccggggatc ctctagagat ccctcgacct cgaccc - #acgc gtccgcccac
    50
            gcgtccgccc acgcgtccgg ggcgtcccag atgttg - #tgga actgtccctg 100
           gatctatagc tcttcaccgt ctctactttc ttcctt - #ctaa gagatcctga 150
           aacctctgtc  atg gaa aag tac ca - #c gtg ttg gag atg att 190
                       - #Met Glu Lys Tyr His Val Leu Glu Met Ile
                       - #  1               5 - #                 10
           gga gaa ggc tct ttt ggg agg - #gtg tac aag ggt cga aga 229
           Gly Glu Gly Ser Phe Gly Arg - #Val Tyr Lys Gly Arg Arg
                       - #     15             - #     20
           aaa tac agt gct cag gtc gtg - #gcc ctg aag ttc atc cca 268
           Lys Tyr Ser Ala Gln Val Val - #Ala Leu Lys Phe Ile Pro
                25       - #           30       - #           35
           aaa ttg ggg cgc tca gag aag - #gag ctg agg aat ttg caa 307
           Lys Leu Gly Arg Ser Glu Lys - #Glu Leu Arg Asn Leu Gln
                       - # 40                 - # 45
           cga gag att gaa ata atg cgg - #ggt ctg cgg cat ccc aac 346
           Arg Glu Ile Glu Ile Met Arg - #Gly Leu Arg His Pro Asn
            50           - #       55           - #       60
           att gtg cat atg ctt gac agc - #ttt gaa act gat aaa gag 385
           Ile Val His Met Leu Asp Ser - #Phe Glu Thr Asp Lys Glu
                    65   - #               70   - #               75
           gtg gtg gtg gtg aca gac tat - #gct gag gga gag ctc ttt 424
           Val Val Val Val Thr Asp Tyr - #Ala Glu Gly Glu Leu Phe
                       - #     80             - #     85
           cag atc cta gaa gat gac gga - #aaa ctt cct gaa gac cag 463
           Gln Ile Leu Glu Asp Asp Gly - #Lys Leu Pro Glu Asp Gln
                90       - #           95       - #          100
           gtt cag gcc att gct gcc cag - #ttg gtg tca gcc ctg tac 502
           Val Gln Ala Ile Ala Ala Gln - #Leu Val Ser Ala Leu Tyr
                       - #105                 - #110
           tat ctg cat tcc cac cgc atc - #cta cac cga gat atg aag 541
           Tyr Leu His Ser His Arg Ile - #Leu His Arg Asp Met Lys
           115            - #     120            - #     125
           cct cag aac atc ctc ctc gcc - #aag ggt ggt ggc atc aag 580
           Pro Gln Asn Ile Leu Leu Ala - #Lys Gly Gly Gly Ile Lys
                   130    - #             135    - #             140
           ctc tgt gac ttt gga ttt gcc - #cgg gct atg agc acc aat 619
           Leu Cys Asp Phe Gly Phe Ala - #Arg Ala Met Ser Thr Asn
                       - #    145             - #    150
           aca atg gtg ctg aca tcc atc - #aaa ggc aca cca ctc tat 658
           Thr Met Val Leu Thr Ser Ile - #Lys Gly Thr Pro Leu Tyr
               155        - #         160        - #         165
           atg tct cca gag ctg gtg gag - #gag cga cca tac gac cac 697
           Met Ser Pro Glu Leu Val Glu - #Glu Arg Pro Tyr Asp His
                       - #170                 - #175
           aca gcg gac ctc tgg tct gtt - #ggc tgc ata cta tat gaa 736
           Thr Ala Asp Leu Trp Ser Val - #Gly Cys Ile Leu Tyr Glu
           180            - #     185            - #     190
           ctg gca gta ggc acc cct ccc - #ttc tat gct aca agc atc 775
           Leu Ala Val Gly Thr Pro Pro - #Phe Tyr Ala Thr Ser Ile
                   195    - #             200    - #             205
           ttt cag ctg gtc agc ctc att - #ctc aag gac cct gtg cgc 814
           Phe Gln Leu Val Ser Leu Ile - #Leu Lys Asp Pro Val Arg
                       - #    210             - #    215
           tgg ccc tca acc atc agt ccc - #tgc ttt aag aac ttc ctg 853
           Trp Pro Ser Thr Ile Ser Pro - #Cys Phe Lys Asn Phe Leu
               220        - #         225        - #         230
           cag gga ctg ctc acc aaa gac - #cca cgg cag cga ctg tcc 892
           Gln Gly Leu Leu Thr Lys Asp - #Pro Arg Gln Arg Leu Ser
                       - #235                 - #240
           tgg cca gac ctc tta tat cac - #ccc ttt att gct ggt cat 931
           Trp Pro Asp Leu Leu Tyr His - #Pro Phe Ile Ala Gly His
           245            - #     250            - #     255
           gtc acc ata ata act gag cca - #gca ggc cca gat ttg ggg 970
           Val Thr Ile Ile Thr Glu Pro - #Ala Gly Pro Asp Leu Gly
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           acc cca ttc acc agc cgc cta - #ccc cca gaa ctt cag gtc 1009
           Thr Pro Phe Thr Ser Arg Leu - #Pro Pro Glu Leu Gln Val
                       - #    275             - #    280
           cta aag gac gaa cag gcc cat - #cgg ttg gcc ccc aag ggt 1048
           Leu Lys Asp Glu Gln Ala His - #Arg Leu Ala Pro Lys Gly
               285        - #         290        - #         295
           aat cag tct cgc atc ttg act - #cag gcc tat aaa cgc atg 1087
           Asn Gln Ser Arg Ile Leu Thr - #Gln Ala Tyr Lys Arg Met
                       - #300                 - #305
           gct gag gag gcc atg cag aag - #aaa cat cag aac aca gga 1126
           Ala Glu Glu Ala Met Gln Lys - #Lys His Gln Asn Thr Gly
           310            - #     315            - #     320
           cct gcc ctt gag caa gag gac - #aag acc agc aag gtg gct 1165
           Pro Ala Leu Glu Gln Glu Asp - #Lys Thr Ser Lys Val Ala
                   325    - #             330    - #             335
           cct ggc aca gcc cct ctg ccc - #aga ctc ggg gcc act cct 1204
           Pro Gly Thr Ala Pro Leu Pro - #Arg Leu Gly Ala Thr Pro
                       - #    340             - #    345
           cag gaa tca agc ctc ctg gcc - #ggg atc tta gcc tca gaa 1243
           Gln Glu Ser Ser Leu Leu Ala - #Gly Ile Leu Ala Ser Glu
               350        - #         355        - #         360
           ttg aag agc agc tgg gct aaa - #tca ggg act gga gag gtg 1282
           Leu Lys Ser Ser Trp Ala Lys - #Ser Gly Thr Gly Glu Val
                       - #365                 - #370
           ccc tct gca cct cgg gaa aac - #cgg acc acc cca gat tgt 1321
           Pro Ser Ala Pro Arg Glu Asn - #Arg Thr Thr Pro Asp Cys
           375            - #     380            - #     385
           gaa cga gca ttc cca gag gag - #agg cca gag gtg ctg ggc 1360
           Glu Arg Ala Phe Pro Glu Glu - #Arg Pro Glu Val Leu Gly
                   390    - #             395    - #             400
           cag cgg agc act gat gta gtg - #gac ctg gaa aat gag gag 1399
           Gln Arg Ser Thr Asp Val Val - #Asp Leu Glu Asn Glu Glu
                       - #    405             - #    410
           cca gac agt gac aat gag tgg - #cag cac ctg cta gag acc 1438
           Pro Asp Ser Asp Asn Glu Trp - #Gln His Leu Leu Glu Thr
               415        - #         420        - #         425
           act gag cct gtg cct att caa - #ctg aag gct cct ctc acc 1477
           Thr Glu Pro Val Pro Ile Gln - #Leu Lys Ala Pro Leu Thr
                       - #430                 - #435
           ttg ctg tgt aat cct gac ttc - #tgc cag cgc atc cag agt 1516
           Leu Leu Cys Asn Pro Asp Phe - #Cys Gln Arg Ile Gln Ser
           440            - #     445            - #     450
           cag ctg cat gaa gct gga ggg - #cag atc ctg aaa ggc atc 1555
           Gln Leu His Glu Ala Gly Gly - #Gln Ile Leu Lys Gly Ile
                   455    - #             460    - #             465
           ttg gag ggt gct tcc cac atc - #ctg cct gca ttc cgg gtc 1594
           Leu Glu Gly Ala Ser His Ile - #Leu Pro Ala Phe Arg Val
                       - #    470             - #    475
           ctg agc agt ctt ctc tcc agc - #tgc agt gat tct gtt gcc 1633
           Leu Ser Ser Leu Leu Ser Ser - #Cys Ser Asp Ser Val Ala
               480        - #         485        - #         490
           ttg tat tcc ttc tgc cgg gag - #gca ggg ctt cct ggg ctg 1672
           Leu Tyr Ser Phe Cys Arg Glu - #Ala Gly Leu Pro Gly Leu
                       - #495                 - #500
           ctg ctg agt cta ctc agg cac - #agt cag gag agc aac agc 1711
           Leu Leu Ser Leu Leu Arg His - #Ser Gln Glu Ser Asn Ser
           505            - #     510            - #     515
           ctc cag cag caa tct tgg tat - #ggg acc ttc tta cag gac 1750
           Leu Gln Gln Gln Ser Trp Tyr - #Gly Thr Phe Leu Gln Asp
                   520    - #             525    - #             530
           ctg atg gct gtg att cag gcc - #tac ttt gcc tgt acc ttc 1789
           Leu Met Ala Val Ile Gln Ala - #Tyr Phe Ala Cys Thr Phe
                       - #    535             - #    540
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           Asn Leu Glu Arg Ser Gln Thr - #Ser Asp Ser Leu Gln Val
               545        - #         550        - #         555
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           Phe Gln Glu Ala Ala Asn Leu - #Phe Leu Asp Leu Leu Gly
                       - #560                 - #565
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           Lys Leu Leu Ala Gln Pro Asp - #Asp Ser Glu Gln Thr Leu
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           Arg Arg Asp Ser Leu Met Cys - #Phe Thr Val Leu Cys Glu
                   585    - #             590    - #             595
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           Ala Met Asp Gly Asn Ser Arg - #Ala Ile Ser Lys Ala Phe
                       - #    600             - #    605
           tac tcc agc ttg ctg acg aca - #cag cag gtt gtc ttg gat 2023
           Tyr Ser Ser Leu Leu Thr Thr - #Gln Gln Val Val Leu Asp
               610        - #         615        - #         620
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           Gly Leu Leu His Gly Leu Thr - #Val Pro Gln Leu Pro Val
                       - #625                 - #630
           cac act ccc caa gga gcc ccg - #caa gtg agc cag cca ctg 2101
           His Thr Pro Gln Gly Ala Pro - #Gln Val Ser Gln Pro Leu
           635            - #     640            - #     645
           cga gag cag agt gag gat ata - #cct gga gcc att tcc tct 2140
           Arg Glu Gln Ser Glu Asp Ile - #Pro Gly Ala Ile Ser Ser
                   650    - #             655    - #             660
           gcc ctg gca gcc ata tgc act - #gct cct gtg gga ctg ccc 2179
           Ala Leu Ala Ala Ile Cys Thr - #Ala Pro Val Gly Leu Pro
                       - #    665             - #    670
           gac tgc tgg gat gcc aag gag - #cag gtc tgt tgg cat ttg 2218
           Asp Cys Trp Asp Ala Lys Glu - #Gln Val Cys Trp His Leu
               675        - #         680        - #         685
           gca aat cag cta act gaa gac - #agc agc cag ctc agg cca 2257
           Ala Asn Gln Leu Thr Glu Asp - #Ser Ser Gln Leu Arg Pro
                       - #690                 - #695
           tcc ctc atc tct ggc ctg cag - #cat ccc atc ctg tgc ctg 2296
           Ser Leu Ile Ser Gly Leu Gln - #His Pro Ile Leu Cys Leu
           700            - #     705            - #     710
           cac ctt ctc aag gtt cta tac - #tcc tgc tgc ctt gtc agt 2335
           His Leu Leu Lys Val Leu Tyr - #Ser Cys Cys Leu Val Ser
                   715    - #             720    - #             725
           gag ggc ctg tgc cgt ctt ctg - #ggg cag gag ccc ctg gcc 2374
           Glu Gly Leu Cys Arg Leu Leu - #Gly Gln Glu Pro Leu Ala
                       - #    730             - #    735
           ttg gaa tcc ctg ttt atg ttg - #att cag ggc aag gta aaa 2413
           Leu Glu Ser Leu Phe Met Leu - #Ile Gln Gly Lys Val Lys
               740        - #         745        - #         750
           gta gta gat tgg gaa gag tct - #act gaa gtg aca ctc tac 2452
           Val Val Asp Trp Glu Glu Ser - #Thr Glu Val Thr Leu Tyr
                       - #755                 - #760
           ttc ctc tcc ctt ctt gtc ttt - #cgg ctc caa aac ctg cct 2491
           Phe Leu Ser Leu Leu Val Phe - #Arg Leu Gln Asn Leu Pro
           765            - #     770            - #     775
           tgt gga atg gag aag cta ggc - #agt gac gtt gct act ctc 2530
           Cys Gly Met Glu Lys Leu Gly - #Ser Asp Val Ala Thr Leu
                   780    - #             785    - #             790
           ttt acc cat tcg cat gtc gtc - #tct ctt gtg agt gca gca 2569
           Phe Thr His Ser His Val Val - #Ser Leu Val Ser Ala Ala
                       - #    795             - #    800
           gcc tgt cta ttg gga cag ctt - #ggt cag caa ggg gtg acc 2608
           Ala Cys Leu Leu Gly Gln Leu - #Gly Gln Gln Gly Val Thr
               805        - #         810        - #         815
           ttt gac ctc cag ccc atg gaa - #tgg atg gct gca gcc aca 2647
           Phe Asp Leu Gln Pro Met Glu - #Trp Met Ala Ala Ala Thr
                       - #820                 - #825
           cat gcc ttg tct gcc cct gca - #gag gtt cgg ttg act cca 2686
           His Ala Leu Ser Ala Pro Ala - #Glu Val Arg Leu Thr Pro
           830            - #     835            - #     840
           cca ggt agt tgt gga ttc tat - #gat ggc ctc ctt atc ctt 2725
           Pro Gly Ser Cys Gly Phe Tyr - #Asp Gly Leu Leu Ile Leu
                   845    - #             850    - #             855
           ctg ttg cag ctc ctc act gag - #cag ggg aag gct agc cta 2764
           Leu Leu Gln Leu Leu Thr Glu - #Gln Gly Lys Ala Ser Leu
                       - #    860             - #    865
           atc agg gat atg tcc agt tca - #gaa atg tgg acc gtt ttg 2803
           Ile Arg Asp Met Ser Ser Ser - #Glu Met Trp Thr Val Leu
               870        - #         875        - #         880
           tgg cac cgc ttc tcc atg gtc - #ctg agg ctc ccc gag gag 2842
           Trp His Arg Phe Ser Met Val - #Leu Arg Leu Pro Glu Glu
                       - #885                 - #890
           gca tct gca cag gaa ggg gag - #ctt tcg cta tcc agt cca 2881
           Ala Ser Ala Gln Glu Gly Glu - #Leu Ser Leu Ser Ser Pro
           895            - #     900            - #     905
           cca agc cct gag cca gac tgg - #aca ctg att tct ccc cag 2920
           Pro Ser Pro Glu Pro Asp Trp - #Thr Leu Ile Ser Pro Gln
                   910    - #             915    - #             920
           ggc atg gca gcc ctg ctg agc - #ctg gcc atg gcc acc ttt 2959
           Gly Met Ala Ala Leu Leu Ser - #Leu Ala Met Ala Thr Phe
                       - #    925             - #    930
           acc cag gag ccc cag tta tgc - #ctg agc tgc ctg tcc cag 2998
           Thr Gln Glu Pro Gln Leu Cys - #Leu Ser Cys Leu Ser Gln
               935        - #         940        - #         945
           cat gga agt atc ctc atg tcc - #atc ctg aag cat ctg ctt 3037
           His Gly Ser Ile Leu Met Ser - #Ile Leu Lys His Leu Leu
                       - #950                 - #955
           tgc ccc agc ttc ctg aat caa - #ctg cgc cag gcg cct cat 3076
           Cys Pro Ser Phe Leu Asn Gln - #Leu Arg Gln Ala Pro His
           960            - #     965            - #     970
           ggg tct gag ttt ctc cct gtc - #gtg gtg ctc tct gtc tgc 3115
           Gly Ser Glu Phe Leu Pro Val - #Val Val Leu Ser Val Cys
                   975    - #             980    - #             985
           cag ctc ctt tgc ttc ccc ttt - #gcg ctg gac atg gat gct 3154
           Gln Leu Leu Cys Phe Pro Phe - #Ala Leu Asp Met Asp Ala
                       - #    990             - #    995
           gac ctc ctt ata gtt gtc ttg - #gcc gac ctc agg gac tca 3193
           Asp Leu Leu Ile Val Val Leu - #Ala Asp Leu Arg Asp Ser
              1000        - #        1005         - #       1010
           gaa gtt gca gcc cat ctg ctg - #cag gtc tgc tgc tac cat 3232
           Glu Val Ala Ala His Leu Leu - #Gln Val Cys Cys Tyr His
                      1015 - #                1020
           ctt ccg ttg atg caa gtg gag - #ctg ccc atc agc ctt ctc 3271
           Leu Pro Leu Met Gln Val Glu - #Leu Pro Ile Ser Leu Leu



           1025           - #     1030            - #    1035
           aca cgc ctg gcc ctc atg gat - #ccc acc tct ctc aac cag 3310
           Thr Arg Leu Ala Leu Met Asp - #Pro Thr Ser Leu Asn Gln
                  1040    - #            1045     - #           1050
           ttt gtg aac aca gtg tct gcc - #tcc cct aga acc atc gtc 3349
           Phe Val Asn Thr Val Ser Ala - #Ser Pro Arg Thr Ile Val
                       - #   1055              - #  1060
           tcg ttt ctc tca gtt gcc ctc - #ctg agt gac cag cca ctg 3388
           Ser Phe Leu Ser Val Ala Leu - #Leu Ser Asp Gln Pro Leu
              1065        - #        1070         - #       1075
           ttg acc tcc gac ctt ctc tct - #ctg ctg gcc cat act gcc 3427
           Leu Thr Ser Asp Leu Leu Ser - #Leu Leu Ala His Thr Ala
                      1080 - #                1085
           agg gtc ctg tct ccc agc cac - #ttg tcc ttt atc caa gag 3466
           Arg Val Leu Ser Pro Ser His - #Leu Ser Phe Ile Gln Glu
           1090           - #     1095            - #    1100
           ctt ctg gct ggc tct gat gaa - #tcc tat cgg ccc ctg cgc 3505
           Leu Leu Ala Gly Ser Asp Glu - #Ser Tyr Arg Pro Leu Arg
                  1105    - #            1110     - #           1115
           agc ctc ctg ggc cac cca gag - #aat tct gtg cgg gca cac 3544
           Ser Leu Leu Gly His Pro Glu - #Asn Ser Val Arg Ala His
                       - #   1120              - #  1125
           act tat agg ctc ctg gga cac - #ttg ctc caa cac agc atg 3583
           Thr Tyr Arg Leu Leu Gly His - #Leu Leu Gln His Ser Met
              1130        - #        1135         - #       1140
           gcc ctg cgt ggg gca ctg cag - #agc cag tct gga ctg ctc 3622
           Ala Leu Arg Gly Ala Leu Gln - #Ser Gln Ser Gly Leu Leu
                      1145 - #                1150
           agc ctt ctg ctg ctt ggg ctt - #gga gac aag gat cct gtt 3661
           Ser Leu Leu Leu Leu Gly Leu - #Gly Asp Lys Asp Pro Val
           1155           - #     1160            - #    1165
           gtg cgg tgc agt gcc agc ttt - #gct gtg ggc aat gca gcc 3700
           Val Arg Cys Ser Ala Ser Phe - #Ala Val Gly Asn Ala Ala
                  1170    - #            1175     - #           1180
           tac cag gct ggt cct ctg gga - #cct gcc ctg gca gct gca 3739
           Tyr Gln Ala Gly Pro Leu Gly - #Pro Ala Leu Ala Ala Ala
                       - #   1185              - #  1190
           gtg ccc agt atg acc cag ctg - #ctt gga gat cct cag gct 3778
           Val Pro Ser Met Thr Gln Leu - #Leu Gly Asp Pro Gln Ala
              1195        - #        1200         - #       1205
           ggt atc cgg cgc aat gtt gca - #tca gct ctg ggc aac ttg 3817
           Gly Ile Arg Arg Asn Val Ala - #Ser Ala Leu Gly Asn Leu
                      1210 - #                1215
           gga cct gaa ggt ttg gga gag - #gag ctg tta cag tgc gaa 3856
           Gly Pro Glu Gly Leu Gly Glu - #Glu Leu Leu Gln Cys Glu
           1220           - #     1225            - #    1230
           gta ccc cag cgg ctc cta gaa - #atg gca tgt gga gac ccc 3895
           Val Pro Gln Arg Leu Leu Glu - #Met Ala Cys Gly Asp Pro
                  1235    - #            1240     - #           1245
           cag cca aat gtg aag gag gct - #gcc ctc att gcc ctc cgg 3934
           Gln Pro Asn Val Lys Glu Ala - #Ala Leu Ile Ala Leu Arg
                       - #   1250              - #  1255
           agc ctg caa cag gag cct ggc - #atc cat cag gta ctg gtg 3973
           Ser Leu Gln Gln Glu Pro Gly - #Ile His Gln Val Leu Val
              1260        - #        1265         - #       1270
           tcc ctg ggt gcc agt gag aaa - #cta tcc ttg ctc tct ctg 4012
           Ser Leu Gly Ala Ser Glu Lys - #Leu Ser Leu Leu Ser Leu
                      1275 - #                1280
           ggg aat cag tca ctg cca cac - #agc agt cct agg cct gcc 4051
           Gly Asn Gln Ser Leu Pro His - #Ser Ser Pro Arg Pro Ala
           1285           - #     1290            - #    1295
           tct gcc aaa cac tgc agg aaa - #ctc att cac ctc ctg agg 4090
           Ser Ala Lys His Cys Arg Lys - #Leu Ile His Leu Leu Arg
                  1300    - #            1305     - #           1310
           cca gcc cat agc atg tgatt ccag - #attcct gcggtccagc 4130
           Pro Ala His Ser Met
                       - #   1315
           ctccaacttt ggtgccagct ctttcttatn taatac - #acaa gcgccaaytc 4180
           aactgagagc taaagagact agaaaagaga taagct - #gcca actcaactga 4230
           gaacaggaaa ctngaagaga tttatatata aagctt - #cttc cttctcccag 4280
           atgcaggatg ttttcaacca gtaaatttta ttgctg - #ttgg tgccagagaa 4330
           gagtcccttt cttctctaca tccaggggcc nttttc - #tcca ataatgtgcc 4380
           tttaactcta gggacctgcc tcacggacct taggga - #aaaa cctcaacctg 4430
           aaagatctct tcctttctgg agctccttta atcttc - #ccag caggtttttg 4480
           ccttagacgt gctggcccca ggacagtgat gaagac - #agag cctgtctcag 4530
           ctctaggctg tggggatcaa tgccatcagt ccctgt - #tatt gagggattat 4580
           cccttagcca acattcctat ctgtgggtgg gcgtgg - #agag tgtatctttt 4630
           tttggggtgt gtgtgtatat gtgtgtgtgt atgtgt - #gtgt gtgtttaata 4680
           gttctgtttg taaactcttt taataaaagt tgtgcc - #tcac catacttgaa 4730
           gctcccagga caagggttga gaggctcaac ccctct - #ttca gcttctatgt 4780
           ggtgttggag gtgctggtat cgtgttcaca caaaaa - #aaaa aaaaaaaaaa 4830
           aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa - #aaaa aaaaaaaaaa 4880
      - -  - - <210> SEQ ID NO 2
     <211> LENGTH: 1315
     <212> TYPE: PRT
     <213> ORGANISM: Homo sapiens
      - - <400> SEQUENCE: 2
      - -       Met Glu Lys Tyr His Val Leu - #Glu Met Ile Gly Glu Gly Ser
    Phe
              1          - #     5             - #     10             - #
    15
           Gly Arg Val Tyr Lys Gly Arg - #Arg Lys Tyr Ser Ala Gln Val Val
                       - #     20             - #     25             - #
    30
            Ala Leu Lys Phe Ile Pro Lys - #Leu Gly Arg Ser Glu Lys Glu Leu
                       - #     35             - #     40             - #
    45
            Arg Asn Leu Gln Arg Glu Ile - #Glu Ile Met Arg Gly Leu Arg His
                       - #     50             - #     55             - #
    60
            Pro Asn Ile Val His Met Leu - #Asp Ser Phe Glu Thr Asp Lys Glu
                       - #     65             - #     70             - #
    75
            Val Val Val Val Thr Asp Tyr - #Ala Glu Gly Glu Leu Phe Gln Ile
                       - #     80             - #     85             - #
    90
            Leu Glu Asp Asp Gly Lys Leu - #Pro Glu Asp Gln Val Gln Ala Ile
                       - #     95             - #    100             - #
    105
            Ala Ala Gln Leu Val Ser Ala - #Leu Tyr Tyr Leu His Ser His Arg
                       - #    110             - #    115             - #
    120
            Ile Leu His Arg Asp Met Lys - #Pro Gln Asn Ile Leu Leu Ala Lys
                       - #    125             - #    130             - #
    135
            Gly Gly Gly Ile Lys Leu Cys - #Asp Phe Gly Phe Ala Arg Ala Met
                       - #    140             - #    145             - #
    150
            Ser Thr Asn Thr Met Val Leu - #Thr Ser Ile Lys Gly Thr Pro Leu
                       - #    155             - #    160             - #
    165
            Tyr Met Ser Pro Glu Leu Val - #Glu Glu Arg Pro Tyr Asp His Thr
                       - #    170             - #    175             - #
    180
            Ala Asp Leu Trp Ser Val Gly - #Cys Ile Leu Tyr Glu Leu Ala Val
                       - #    185             - #    190             - #
    195
            Gly Thr Pro Pro Phe Tyr Ala - #Thr Ser Ile Phe Gln Leu Val Ser
                       - #    200             - #    205             - #
    210
            Leu Ile Leu Lys Asp Pro Val - #Arg Trp Pro Ser Thr Ile Ser Pro
                       - #    215             - #    220             - #
    225
            Cys Phe Lys Asn Phe Leu Gln - #Gly Leu Leu Thr Lys Asp Pro Arg
                       - #    230             - #    235             - #
    240
            Gln Arg Leu Ser Trp Pro Asp - #Leu Leu Tyr His Pro Phe Ile Ala
                       - #    245             - #    250             - #
    255
            Gly His Val Thr Ile Ile Thr - #Glu Pro Ala Gly Pro Asp Leu Gly
                       - #    260             - #    265             - #
    270
            Thr Pro Phe Thr Ser Arg Leu - #Pro Pro Glu Leu Gln Val Leu Lys
                       - #    275             - #    280             - #
    285
            Asp Glu Gln Ala His Arg Leu - #Ala Pro Lys Gly Asn Gln Ser Arg
                       - #    290             - #    295             - #
    300
            Ile Leu Thr Gln Ala Tyr Lys - #Arg Met Ala Glu Glu Ala Met Gln
                       - #    305             - #    310             - #
    315
            Lys Lys His Gln Asn Thr Gly - #Pro Ala Leu Glu Gln Glu Asp Lys
                       - #    320             - #    325             - #
    330
            Thr Ser Lys Val Ala Pro Gly - #Thr Ala Pro Leu Pro Arg Leu Gly
                       - #    335             - #    340             - #
    345
            Ala Thr Pro Gln Glu Ser Ser - #Leu Leu Ala Gly Ile Leu Ala Ser
                       - #    350             - #    355             - #
    360
            Glu Leu Lys Ser Ser Trp Ala - #Lys Ser Gly Thr Gly Glu Val Pro
                       - #    365             - #    370             - #
    375
            Ser Ala Pro Arg Glu Asn Arg - #Thr Thr Pro Asp Cys Glu Arg Ala
                       - #    380             - #    385             - #
    390
            Phe Pro Glu Glu Arg Pro Glu - #Val Leu Gly Gln Arg Ser Thr Asp
                       - #    395             - #    400             - #
    405
            Val Val Asp Leu Glu Asn Glu - #Glu Pro Asp Ser Asp Asn Glu Trp
                       - #    410             - #    415             - #
    420
            Gln His Leu Leu Glu Thr Thr - #Glu Pro Val Pro Ile Gln Leu Lys
                       - #    425             - #    430             - #
    435
            Ala Pro Leu Thr Leu Leu Cys - #Asn Pro Asp Phe Cys Gln Arg Ile
                       - #    440             - #    445             - #
    450
            Gln Ser Gln Leu His Glu Ala - #Gly Gly Gln Ile Leu Lys Gly Ile
                       - #    455             - #    460             - #
    465
            Leu Glu Gly Ala Ser His Ile - #Leu Pro Ala Phe Arg Val Leu Ser
                       - #    470             - #    475             - #
    480
            Ser Leu Leu Ser Ser Cys Ser - #Asp Ser Val Ala Leu Tyr Ser Phe
                       - #    485             - #    490             - #
    495
            Cys Arg Glu Ala Gly Leu Pro - #Gly Leu Leu Leu Ser Leu Leu Arg
                       - #    500             - #    505             - #
    510
            His Ser Gln Glu Ser Asn Ser - #Leu Gln Gln Gln Ser Trp Tyr Gly
                       - #    515             - #    520             - #
    525
            Thr Phe Leu Gln Asp Leu Met - #Ala Val Ile Gln Ala Tyr Phe Ala
                       - #    530             - #    535             - #
    540
            Cys Thr Phe Asn Leu Glu Arg - #Ser Gln Thr Ser Asp Ser Leu Gln
                       - #    545             - #    550             - #
    555
            Val Phe Gln Glu Ala Ala Asn - #Leu Phe Leu Asp Leu Leu Gly Lys
                       - #    560             - #    565             - #
    570
            Leu Leu Ala Gln Pro Asp Asp - #Ser Glu Gln Thr Leu Arg Arg Asp
                       - #    575             - #    580             - #
    585
            Ser Leu Met Cys Phe Thr Val - #Leu Cys Glu Ala Met Asp Gly Asn
                       - #    590             - #    595             - #
    600
            Ser Arg Ala Ile Ser Lys Ala - #Phe Tyr Ser Ser Leu Leu Thr Thr
                       - #    605             - #    610             - #
    615
            Gln Gln Val Val Leu Asp Gly - #Leu Leu His Gly Leu Thr Val Pro
                       - #    620             - #    625             - #
    630
            Gln Leu Pro Val His Thr Pro - #Gln Gly Ala Pro Gln Val Ser Gln
                       - #    635             - #    640             - #
    645
            Pro Leu Arg Glu Gln Ser Glu - #Asp Ile Pro Gly Ala Ile Ser Ser
                       - #    650             - #    655             - #
    660
            Ala Leu Ala Ala Ile Cys Thr - #Ala Pro Val Gly Leu Pro Asp Cys
                       - #    665             - #    670             - #
    675
            Trp Asp Ala Lys Glu Gln Val - #Cys Trp His Leu Ala Asn Gln Leu
                       - #    680             - #    685             - #
    690
            Thr Glu Asp Ser Ser Gln Leu - #Arg Pro Ser Leu Ile Ser Gly Leu
                       - #    695             - #    700             - #
    705
            Gln His Pro Ile Leu Cys Leu - #His Leu Leu Lys Val Leu Tyr Ser
                       - #    710             - #    715             - #
    720
            Cys Cys Leu Val Ser Glu Gly - #Leu Cys Arg Leu Leu Gly Gln Glu
                       - #    725             - #    730             - #
    735
            Pro Leu Ala Leu Glu Ser Leu - #Phe Met Leu Ile Gln Gly Lys Val
                       - #    740             - #    745             - #
    750
            Lys Val Val Asp Trp Glu Glu - #Ser Thr Glu Val Thr Leu Tyr Phe
                       - #    755             - #    760             - #
    765
            Leu Ser Leu Leu Val Phe Arg - #Leu Gln Asn Leu Pro Cys Gly Met
                       - #    770             - #    775             - #
    780
            Glu Lys Leu Gly Ser Asp Val - #Ala Thr Leu Phe Thr His Ser His
                       - #    785             - #    790             - #
    795
            Val Val Ser Leu Val Ser Ala - #Ala Ala Cys Leu Leu Gly Gln Leu
                       - #    800             - #    805             - #
    810
            Gly Gln Gln Gly Val Thr Phe - #Asp Leu Gln Pro Met Glu Trp Met
                       - #    815             - #    820             - #
    825
            Ala Ala Ala Thr His Ala Leu - #Ser Ala Pro Ala Glu Val Arg Leu
                       - #    830             - #    835             - #



    840
            Thr Pro Pro Gly Ser Cys Gly - #Phe Tyr Asp Gly Leu Leu Ile Leu
                       - #    845             - #    850             - #
    855
            Leu Leu Gln Leu Leu Thr Glu - #Gln Gly Lys Ala Ser Leu Ile Arg
                       - #    860             - #    865             - #
    870
            Asp Met Ser Ser Ser Glu Met - #Trp Thr Val Leu Trp His Arg Phe
                       - #    875             - #    880             - #
    885
            Ser Met Val Leu Arg Leu Pro - #Glu Glu Ala Ser Ala Gln Glu Gly
                       - #    890             - #    895             - #
    900
            Glu Leu Ser Leu Ser Ser Pro - #Pro Ser Pro Glu Pro Asp Trp Thr
                       - #    905             - #    910             - #
    915
            Leu Ile Ser Pro Gln Gly Met - #Ala Ala Leu Leu Ser Leu Ala Met
                       - #    920             - #    925             - #
    930
            Ala Thr Phe Thr Gln Glu Pro - #Gln Leu Cys Leu Ser Cys Leu Ser
                       - #    935             - #    940             - #
    945
            Gln His Gly Ser Ile Leu Met - #Ser Ile Leu Lys His Leu Leu Cys
                       - #    950             - #    955             - #
    960
            Pro Ser Phe Leu Asn Gln Leu - #Arg Gln Ala Pro His Gly Ser Glu
                       - #    965             - #    970             - #
    975
            Phe Leu Pro Val Val Val Leu - #Ser Val Cys Gln Leu Leu Cys Phe
                       - #    980             - #    985             - #
    990
            Pro Phe Ala Leu Asp Met Asp - #Ala Asp Leu Leu Ile Val Val Leu
                       - #    995             - #   1000              - #
    1005
            Ala Asp Leu Arg Asp Ser Glu - #Val Ala Ala His Leu Leu Gln Val
                       - #   1010              - #  1015               - #
    1020
            Cys Cys Tyr His Leu Pro Leu - #Met Gln Val Glu Leu Pro Ile Ser
                       - #   1025              - #  1030               - #
    1035
            Leu Leu Thr Arg Leu Ala Leu - #Met Asp Pro Thr Ser Leu Asn Gln
                       - #   1040              - #  1045               - #
    1050
            Phe Val Asn Thr Val Ser Ala - #Ser Pro Arg Thr Ile Val Ser Phe
                       - #   1055              - #  1060               - #
    1065
            Leu Ser Val Ala Leu Leu Ser - #Asp Gln Pro Leu Leu Thr Ser Asp
                       - #   1070              - #  1075               - #
    1080
            Leu Leu Ser Leu Leu Ala His - #Thr Ala Arg Val Leu Ser Pro Ser
                       - #   1085              - #  1090               - #
    1095
            His Leu Ser Phe Ile Gln Glu - #Leu Leu Ala Gly Ser Asp Glu Ser
                       - #   1100              - #  1105               - #
    1110
            Tyr Arg Pro Leu Arg Ser Leu - #Leu Gly His Pro Glu Asn Ser Val
                       - #   1115              - #  1120               - #
    1125
            Arg Ala His Thr Tyr Arg Leu - #Leu Gly His Leu Leu Gln His Ser
                       - #   1130              - #  1135               - #
    1140
            Met Ala Leu Arg Gly Ala Leu - #Gln Ser Gln Ser Gly Leu Leu Ser
                       - #   1145              - #  1150               - #
    1155
            Leu Leu Leu Leu Gly Leu Gly - #Asp Lys Asp Pro Val Val Arg Cys
                       - #   1160              - #  1165               - #
    1170
            Ser Ala Ser Phe Ala Val Gly - #Asn Ala Ala Tyr Gln Ala Gly Pro
                       - #   1175              - #  1180               - #
    1185
            Leu Gly Pro Ala Leu Ala Ala - #Ala Val Pro Ser Met Thr Gln Leu
                       - #   1190              - #  1195               - #
    1200
            Leu Gly Asp Pro Gln Ala Gly - #Ile Arg Arg Asn Val Ala Ser Ala
                       - #   1205              - #  1210               - #
    1215
            Leu Gly Asn Leu Gly Pro Glu - #Gly Leu Gly Glu Glu Leu Leu Gln
                       - #   1220              - #  1225               - #
    1230
            Cys Glu Val Pro Gln Arg Leu - #Leu Glu Met Ala Cys Gly Asp Pro
                       - #   1235              - #  1240               - #
    1245
            Gln Pro Asn Val Lys Glu Ala - #Ala Leu Ile Ala Leu Arg Ser Leu
                       - #   1250              - #  1255               - #
    1260
            Gln Gln Glu Pro Gly Ile His - #Gln Val Leu Val Ser Leu Gly Ala
                       - #   1265              - #  1270               - #
    1275
            Ser Glu Lys Leu Ser Leu Leu - #Ser Leu Gly Asn Gln Ser Leu Pro
                       - #   1280              - #  1285               - #
    1290
            His Ser Ser Pro Arg Pro Ala - #Ser Ala Lys His Cys Arg Lys Leu
                       - #   1295              - #  1300               - #
    1305
            Ile His Leu Leu Arg Pro Ala - #His Ser Met
                        - #   1310              - #  1315
       - -  - - <210> SEQ ID NO 3
      <211> LENGTH: 193
      <212> TYPE: DNA
      <213> ORGANISM: Homo sapiens
       - - <400> SEQUENCE: 3
       - -       cccgggctat gagcaccaat acaatggtgc tgacat - #ccat caaaggcaca
    50
            ccactctata tgtctccaga gctggtggag gagcga - #ccat acgaccacac 100
           agcggacctc tggtctgttg gctgcatact atatga - #actg gcagtaggca 150
           cccctccctt ctaatgctac aagcatcttt cagctg - #gtca gcc 193
      - -  - - <210> SEQ ID NO 4
     <211> LENGTH: 5125
     <212> TYPE: DNA
     <213> ORGANISM: Homo sapiens
      - - <400> SEQUENCE: 4
      - -       cccacgcgtc cgcccacgcg tccggggcgt cccaga - #tgtt gtggaactgt 50
           ccctggatct atagctcttc accgtctcta ctttct - #tcct tctaagagat 100
           cctgaaacct ctgtc    atg gaa - #aag tac cac gtg ttg gag 139
                       - #        Met Glu Lys Tyr His Val - #Leu Glu
                       - #          1        - #       5
           atg att gga gaa ggc tct ttt - #ggg agg gtg tac aag ggt 178
           Met Ile Gly Glu Gly Ser Phe - #Gly Arg Val Tyr Lys Gly
                10       - #           15       - #           20
           cga aga aaa tac agt gct cag - #gtc gtg gcc ctg aag ttc 217
           Arg Arg Lys Tyr Ser Ala Gln - #Val Val Ala Leu Lys Phe
                       - # 25                 - # 30
           atc cca aaa ttg ggg cgc tca - #gag aag gag ctg agg aat 256
           Ile Pro Lys Leu Gly Arg Ser - #Glu Lys Glu Leu Arg Asn
            35           - #       40           - #       45
           ttg caa cga gag att gaa ata - #atg cgg ggt ctg cgg cat 295
           Leu Gln Arg Glu Ile Glu Ile - #Met Arg Gly Leu Arg His
                    50   - #               55   - #               60
           ccc aac att gtg cat atg ctt - #gac agc ttt gaa act gat 334
           Pro Asn Ile Val His Met Leu - #Asp Ser Phe Glu Thr Asp
                       - #     65             - #     70
           aaa gag gtg gtg gtg gtg aca - #gac tat gct gag gga gag 373
           Lys Glu Val Val Val Val Thr - #Asp Tyr Ala Glu Gly Glu
                75       - #           80       - #           85
           ctc ttt cag atc cta gaa gat - #gac gga aaa ctt cct gaa 412
           Leu Phe Gln Ile Leu Glu Asp - #Asp Gly Lys Leu Pro Glu
                       - # 90                 - # 95
           gac cag gtt cag gcc att gct - #gcc cag ttg gtg tca gcc 451
           Asp Gln Val Gln Ala Ile Ala - #Ala Gln Leu Val Ser Ala
           100            - #     105            - #     110
           ctg tac tat ctg cat tcc cac - #cgc atc cta cac cga gat 490
           Leu Tyr Tyr Leu His Ser His - #Arg Ile Leu His Arg Asp
                   115    - #             120    - #             125
           atg aag cct cag aac atc ctc - #ctc gcc aag ggt ggt ggc 529
           Met Lys Pro Gln Asn Ile Leu - #Leu Ala Lys Gly Gly Gly
                       - #    130             - #    135
           atc aag ctc tgt gac ttt gga - #ttt gcc cgg gct atg agc 568
           Ile Lys Leu Cys Asp Phe Gly - #Phe Ala Arg Ala Met Ser
               140        - #         145        - #         150
           acc aat aca atg gtg ctg aca - #tcc atc aaa ggc aca cca 607
           Thr Asn Thr Met Val Leu Thr - #Ser Ile Lys Gly Thr Pro
                       - #155                 - #160
           ctc tat atg tct cca gag ctg - #gtg gag gag cga cca tac 646
           Leu Tyr Met Ser Pro Glu Leu - #Val Glu Glu Arg Pro Tyr
           165            - #     170            - #     175
           gac cac aca gcg gac ctc tgg - #tct gtt ggc tgc ata cta 685
           Asp His Thr Ala Asp Leu Trp - #Ser Val Gly Cys Ile Leu
                   180    - #             185    - #             190
           tat gaa ctg gca gta ggc acc - #cct ccc ttc tat gct aca 724
           Tyr Glu Leu Ala Val Gly Thr - #Pro Pro Phe Tyr Ala Thr
                       - #    195             - #    200
           agc atc ttt cag ctg gtc agc - #ctc att ctc aag gac cct 763
           Ser Ile Phe Gln Leu Val Ser - #Leu Ile Leu Lys Asp Pro
               205        - #         210        - #         215
           gtg cgc tgg ccc tca acc atc - #agt ccc tgc ttt aag aac 802
           Val Arg Trp Pro Ser Thr Ile - #Ser Pro Cys Phe Lys Asn
                       - #220                 - #225
           ttc ctg cag gga ctg ctc acc - #aaa gac cca cgg cag cga 841
           Phe Leu Gln Gly Leu Leu Thr - #Lys Asp Pro Arg Gln Arg
           230            - #     235            - #     240
           ctg tcc tgg cca gac ctc tta - #tat cac ccc ttt att gct 880
           Leu Ser Trp Pro Asp Leu Leu - #Tyr His Pro Phe Ile Ala
                   245    - #             250    - #             255
           ggt cat gtc acc ata ata act - #gag cca gca ggc cca gat 919
           Gly His Val Thr Ile Ile Thr - #Glu Pro Ala Gly Pro Asp
                       - #    260             - #    265
           ttg ggg acc cca ttc acc agc - #cgc cta ccc cca gaa ctt 958
           Leu Gly Thr Pro Phe Thr Ser - #Arg Leu Pro Pro Glu Leu
               270        - #         275        - #         280
           cag gtc cta aag gac gaa cag - #gcc cat cgg ttg gcc ccc 997
           Gln Val Leu Lys Asp Glu Gln - #Ala His Arg Leu Ala Pro
                       - #285                 - #290
           aag ggt aat cag tct cgc atc - #ttg act cag gcc tat aaa 1036
           Lys Gly Asn Gln Ser Arg Ile - #Leu Thr Gln Ala Tyr Lys
           295            - #     300            - #     305
           cgc atg gct gag gag gcc atg - #cag aag aaa cat cag aac 1075
           Arg Met Ala Glu Glu Ala Met - #Gln Lys Lys His Gln Asn
                   310    - #             315    - #             320
           aca gga cct gcc ctt gag caa - #gag gac aag acc agc aag 1114
           Thr Gly Pro Ala Leu Glu Gln - #Glu Asp Lys Thr Ser Lys
                       - #    325             - #    330
           gtg gct cct ggc aca gcc cct - #ctg ccc aga ctc ggg gcc 1153
           Val Ala Pro Gly Thr Ala Pro - #Leu Pro Arg Leu Gly Ala
               335        - #         340        - #         345
           act cct cag gaa tca agc ctc - #ctg gcc ggg atc tta gcc 1192
           Thr Pro Gln Glu Ser Ser Leu - #Leu Ala Gly Ile Leu Ala
                       - #350                 - #355
           tca gaa ttg aag agc agc tgg - #gct aaa tca ggg act gga 1231
           Ser Glu Leu Lys Ser Ser Trp - #Ala Lys Ser Gly Thr Gly
           360            - #     365            - #     370
           gag gtg ccc tct gca cct cgg - #gaa aac cgg acc acc cca 1270
           Glu Val Pro Ser Ala Pro Arg - #Glu Asn Arg Thr Thr Pro
                   375    - #             380    - #             385
           gat tgt gaa cga gca ttc cca - #gag gag agg cca gag gtg 1309
           Asp Cys Glu Arg Ala Phe Pro - #Glu Glu Arg Pro Glu Val
                       - #    390             - #    395
           ctg ggc cag cgg agc act gat - #gta gtg gac ctg gaa aat 1348
           Leu Gly Gln Arg Ser Thr Asp - #Val Val Asp Leu Glu Asn
               400        - #         405        - #         410
           gag gag cca gac agt gac aat - #gag tgg cag cac ctg cta 1387
           Glu Glu Pro Asp Ser Asp Asn - #Glu Trp Gln His Leu Leu
                       - #415                 - #420
           gag acc act gag cct gtg cct - #att caa ctg aag gct cct 1426
           Glu Thr Thr Glu Pro Val Pro - #Ile Gln Leu Lys Ala Pro
           425            - #     430            - #     435
           ctc acc ttg ctg tgt aat cct - #gac ttc tgc cag cgc atc 1465
           Leu Thr Leu Leu Cys Asn Pro - #Asp Phe Cys Gln Arg Ile
                   440    - #             445    - #             450
           cag agt cag ctg cat gaa gct - #gga ggg cag atc ctg aaa 1504
           Gln Ser Gln Leu His Glu Ala - #Gly Gly Gln Ile Leu Lys
                       - #    455             - #    460
           ggc atc ttg gag ggt gct tcc - #cac atc ctg cct gca ttc 1543
           Gly Ile Leu Glu Gly Ala Ser - #His Ile Leu Pro Ala Phe
               465        - #         470        - #         475
           cgg gtc ctg agc agt ctt ctc - #tcc agc tgc agt gat tct 1582
           Arg Val Leu Ser Ser Leu Leu - #Ser Ser Cys Ser Asp Ser
                       - #480                 - #485
           gtt gcc ttg tat tcc ttc tgc - #cgg gag gca ggg ctt cct 1621
           Val Ala Leu Tyr Ser Phe Cys - #Arg Glu Ala Gly Leu Pro
           490            - #     495            - #     500
           ggg ctg ctg ctg agt cta ctc - #agg cac agt cag gag agc 1660
           Gly Leu Leu Leu Ser Leu Leu - #Arg His Ser Gln Glu Ser
                   505    - #             510    - #             515
           aac agc ctc cag cag caa tct - #tgg tat ggg acc ttc tta 1699
           Asn Ser Leu Gln Gln Gln Ser - #Trp Tyr Gly Thr Phe Leu
                       - #    520             - #    525
           cag gac ctg atg gct gtg att - #cag gcc tac ttt gcc tgt 1738
           Gln Asp Leu Met Ala Val Ile - #Gln Ala Tyr Phe Ala Cys
               530        - #         535        - #         540
           acc ttc aat ctg gag agg agc - #cag aca agt gac agc ctg 1777
           Thr Phe Asn Leu Glu Arg Ser - #Gln Thr Ser Asp Ser Leu
                       - #545                 - #550
           cag gtg ttt cag gag gct gcc - #aac ctt ttt ctg gac ctg 1816
           Gln Val Phe Gln Glu Ala Ala - #Asn Leu Phe Leu Asp Leu
           555            - #     560            - #     565
           ttg ggg aaa ctg ctg gcc caa - #cca gat gac tct gag cag 1855
           Leu Gly Lys Leu Leu Ala Gln - #Pro Asp Asp Ser Glu Gln
                   570    - #             575    - #             580
           act ttg cag agg gac agc ctt - #atg tgc ttt act gtc ctg 1894
           Thr Leu Gln Arg Asp Ser Leu - #Met Cys Phe Thr Val Leu
                       - #    585             - #    590
           tgc gaa gcc atg gat ggg aac - #agc cgg gcc atc tcc aaa 1933
           Cys Glu Ala Met Asp Gly Asn - #Ser Arg Ala Ile Ser Lys
               595        - #         600        - #         605
           gcc ttt tac tcc agc ttg ctg - #acg aca cag cag gtt gtc 1972



           Ala Phe Tyr Ser Ser Leu Leu - #Thr Thr Gln Gln Val Val
                       - #610                 - #615
           ttg gat ggg ctc ctt cat ggc - #ttg aca gtt cca cag ctc 2011
           Leu Asp Gly Leu Leu His Gly - #Leu Thr Val Pro Gln Leu
           620            - #     625            - #     630
           cct gtc cac act ccc caa ggt - #aac cag agt gga gaa ggg 2050
           Pro Val His Thr Pro Gln Gly - #Asn Gln Ser Gly Glu Gly
                   635    - #             640    - #             645
           agg ttc tct t gacttacttg ttgcatag - #gt caggctccgc 2090
           Arg Phe Ser
                   648
           tctttctatt gccatcacct agatcgcacc tggcat - #ttag taggtgctca 2140
           ataaataact gtgaactgag agaatgaatg gggatc - #tgag ggaaacaaac 2190
           agacctcatc ctgcattctt cccactccct taggtt - #ccct actcctgctg 2240
           ccatgtcggt gagtactggt gctattgtct agggca - #agag cctcaggcct 2290
           ttgg    agt tac tct ttg - #ctt ttc tcc aca gga gcc ccg 2327
                   Ser Tyr Se - #r Leu Leu Phe Ser Thr Gly Ala Pro
                     1  - #             5     - #             10
           caa gtg agc cag cca ctg cga - #gag cag agt gag gat ata 2366
           Gln Val Ser Gln Pro Leu Arg - #Glu Gln Ser Glu Asp Ile
                       - # 15                 - # 20
           cct gga gcc att tcc tct gcc - #ctg gca gcc ata tgc act 2405
           Pro Gly Ala Ile Ser Ser Ala - #Leu Ala Ala Ile Cys Thr
            25           - #       30           - #       35
           gct cct gtg gga ctg ccc gac - #tgc tgg gat gcc aag gag 2444
           Ala Pro Val Gly Leu Pro Asp - #Cys Trp Asp Ala Lys Glu
                    40   - #               45   - #               50
           cag gtc tgt tgg cat ttg gca - #aat cag cta act gaa gac 2483
           Gln Val Cys Trp His Leu Ala - #Asn Gln Leu Thr Glu Asp
                       - #     55             - #     60
           agc agc cag ctc agg cca tcc - #ctc atc tct ggc ctg cag 2522
           Ser Ser Gln Leu Arg Pro Ser - #Leu Ile Ser Gly Leu Gln
                65       - #           70       - #           75
           cat ccc atc ctg tgc ctg cac - #ctt ctc aag gtt cta tac 2561
           His Pro Ile Leu Cys Leu His - #Leu Leu Lys Val Leu Tyr
                       - # 80                 - # 85
           tcc tgc tgc ctt gtc agt gag - #ggc ctg tgc cgt ctt ctg 2600
           Ser Cys Cys Leu Val Ser Glu - #Gly Leu Cys Arg Leu Leu
            90           - #       95           - #      100
           ggg cag gag ccc ctg gcc ttg - #gaa tcc ctg ttt atg ttg 2639
           Gly Gln Glu Pro Leu Ala Leu - #Glu Ser Leu Phe Met Leu
                   105    - #             110    - #             115
           att cag ggc aag gta aaa gta - #gta gat tgg gaa gag tct 2678
           Ile Gln Gly Lys Val Lys Val - #Val Asp Trp Glu Glu Ser
                       - #    120             - #    125
           act gaa gtg aca ctc tac ttc - #ctc tcc ctt ctt gtc ttt 2717
           Thr Glu Val Thr Leu Tyr Phe - #Leu Ser Leu Leu Val Phe
               130        - #         135        - #         140
           cgg ctc caa aac ctg cct tgt - #gga atg gag aag cta ggc 2756
           Arg Leu Gln Asn Leu Pro Cys - #Gly Met Glu Lys Leu Gly
                       - #145                 - #150
           agt gac gtt gct act ctc ttt - #acc cat tcg cat gtc gtc 2795
           Ser Asp Val Ala Thr Leu Phe - #Thr His Ser His Val Val
           155            - #     160            - #     165
           tct ctt gtg agt gca gca gcc - #tgt cta ttg gga cag ctt 2834
           Ser Leu Val Ser Ala Ala Ala - #Cys Leu Leu Gly Gln Leu
                   170    - #             175    - #             180
           ggt cag caa ggg gtg acc ttt - #gac ctc cag ccc atg gaa 2873
           Gly Gln Gln Gly Val Thr Phe - #Asp Leu Gln Pro Met Glu
                       - #    185             - #    190
           tgg atg gct gca gcc aca cat - #gcc ttg tct gcc cct gca 2912
           Trp Met Ala Ala Ala Thr His - #Ala Leu Ser Ala Pro Ala
               195        - #         200        - #         205
           gag gtt cgg ttg act cca cca - #ggt agt tgt gga ttc tat 2951
           Glu Val Arg Leu Thr Pro Pro - #Gly Ser Cys Gly Phe Tyr
                       - #210                 - #215
           gat ggc ctc ctt atc ctt ctg - #ttg cag ctc ctc act gag 2990
           Asp Gly Leu Leu Ile Leu Leu - #Leu Gln Leu Leu Thr Glu
           220            - #     225            - #     230
           cag ggg aag gct agc cta atc - #agg gat atg tcc agt tca 3029
           Gln Gly Lys Ala Ser Leu Ile - #Arg Asp Met Ser Ser Ser
                   235    - #             240    - #             245
           gaa atg tgg acc gtt ttg tgg - #cac cgc ttc tcc atg gtc 3068
           Glu Met Trp Thr Val Leu Trp - #His Arg Phe Ser Met Val
                       - #    250             - #    255
           ctg agg ctc ccc gag gag gca - #tct gca cag gaa ggg gag 3107
           Leu Arg Leu Pro Glu Glu Ala - #Ser Ala Gln Glu Gly Glu
               260        - #         265        - #         270
           ctt tcg cta tcc agt cca cca - #agc cct gag cca gac tgg 3146
           Leu Ser Leu Ser Ser Pro Pro - #Ser Pro Glu Pro Asp Trp
                       - #275                 - #280
           aca ctg att tct ccc cag ggc - #atg gca gcc ctg ctg agc 3185
           Thr Leu Ile Ser Pro Gln Gly - #Met Ala Ala Leu Leu Ser
           285            - #     290            - #     295
           ctg gcc atg gcc acc ttt acc - #cag gag ccc cag tta tgc 3224
           Leu Ala Met Ala Thr Phe Thr - #Gln Glu Pro Gln Leu Cys
                   300    - #             305    - #             310
           ctg agc tgc ctg tcc cag cat - #gga agt atc ctc atg tcc 3263
           Leu Ser Cys Leu Ser Gln His - #Gly Ser Ile Leu Met Ser
                       - #    315             - #    320
           atc ctg aag cat ctg ctt tgc - #ccc agc ttc ctg aat caa 3302
           Ile Leu Lys His Leu Leu Cys - #Pro Ser Phe Leu Asn Gln
               325        - #         330        - #         335
           ctg cgc cag gcg cct cat ggg - #tct gag ttt ctc cct gtc 3341
           Leu Arg Gln Ala Pro His Gly - #Ser Glu Phe Leu Pro Val
                       - #340                 - #345
           gtg gtg ctc tct gtc tgc cag - #ctc ctt tgc ttc ccc ttt 3380
           Val Val Leu Ser Val Cys Gln - #Leu Leu Cys Phe Pro Phe
           350            - #     355            - #     360
           gcg ctg gac atg gat gct gac - #ctc ctt ata gtt gtc ttg 3419
           Ala Leu Asp Met Asp Ala Asp - #Leu Leu Ile Val Val Leu
                   365    - #             370    - #             375
           gcc gac ctc agg gac tca gaa - #gtt gca gcc cat ctg ctg 3458
           Ala Asp Leu Arg Asp Ser Glu - #Val Ala Ala His Leu Leu
                       - #    380             - #    385
           cag gtc tgc tgc tac cat ctt - #ccg ttg atg caa gtg gag 3497
           Gln Val Cys Cys Tyr His Leu - #Pro Leu Met Gln Val Glu
               390        - #         395        - #         400
           ctg ccc atc agc ctt ctc aca - #cgc ctg gcc ctc atg gat 3536
           Leu Pro Ile Ser Leu Leu Thr - #Arg Leu Ala Leu Met Asp
                       - #405                 - #410
           ccc acc tct ctc aac cag ttt - #gtg aac aca gtg tct gcc 3575
           Pro Thr Ser Leu Asn Gln Phe - #Val Asn Thr Val Ser Ala
           415            - #     420            - #     425
           tcc cct aga acc atc gtc tcg - #ttt ctc tca gtt gcc ctc 3614
           Ser Pro Arg Thr Ile Val Ser - #Phe Leu Ser Val Ala Leu
                   430    - #             435    - #             440
           ctg agt gac cag cca ctg ttg - #acc tcc gac ctt ctc tct 3653
           Leu Ser Asp Gln Pro Leu Leu - #Thr Ser Asp Leu Leu Ser
                       - #    445             - #    450
           ctg ctg gcc cat act gcc agg - #gtc ctg tct ccc agc cac 3692
           Leu Leu Ala His Thr Ala Arg - #Val Leu Ser Pro Ser His
               455        - #         460        - #         465
           ttg tcc ttt atc caa gag ctt - #ctg gct ggc tct gat gaa 3731
           Leu Ser Phe Ile Gln Glu Leu - #Leu Ala Gly Ser Asp Glu
                       - #470                 - #475
           tcc tat cgg ccc ctg cgc agc - #ctc ctg ggc cac cca gag 3770
           Ser Tyr Arg Pro Leu Arg Ser - #Leu Leu Gly His Pro Glu
           480            - #     485            - #     490
           aat tct gtg cgg gca cac act - #tat agg ctc ctg gga cac 3809
           Asn Ser Val Arg Ala His Thr - #Tyr Arg Leu Leu Gly His
                   495    - #             500    - #             505
           ttg ctc caa cac agc atg gcc - #ctg cgt ggg gca ctg cag 3848
           Leu Leu Gln His Ser Met Ala - #Leu Arg Gly Ala Leu Gln
                       - #    510             - #    515
           agc cag tct gga ctg ctc agc - #ctt ctg ctg ctt ggg ctt 3887
           Ser Gln Ser Gly Leu Leu Ser - #Leu Leu Leu Leu Gly Leu
               520        - #         525        - #         530
           gga gac aag gat cct gtt gtg - #cgg tgc agt gcc agc ttt 3926
           Gly Asp Lys Asp Pro Val Val - #Arg Cys Ser Ala Ser Phe
                       - #535                 - #540
           gct gtg ggc aat gca gcc tac - #cag gct ggt cct ctg gga 3965
           Ala Val Gly Asn Ala Ala Tyr - #Gln Ala Gly Pro Leu Gly
           545            - #     550            - #     555
           cct gcc ctg gca gct gca gtg - #ccc agt atg acc cag ctg 4004
           Pro Ala Leu Ala Ala Ala Val - #Pro Ser Met Thr Gln Leu
                   560    - #             565    - #             570
           ctt gga gat cct cag gct ggt - #atc cgg cgc aat gtt gca 4043
           Leu Gly Asp Pro Gln Ala Gly - #Ile Arg Arg Asn Val Ala
                       - #    575             - #    580
           tca gct ctg ggc aac ttg gga - #cct gaa ggt ttg gga gag 4082
           Ser Ala Leu Gly Asn Leu Gly - #Pro Glu Gly Leu Gly Glu
               585        - #         590        - #         595
           gag ctg tta cag tgc gaa gta - #ccc cag cgg ctc cta gaa 4121
           Glu Leu Leu Gln Cys Glu Val - #Pro Gln Arg Leu Leu Glu
                       - #600                 - #605
           atg gca tgt gga gac ccc cag - #cca aat gtg aag gag gct 4160
           Met Ala Cys Gly Asp Pro Gln - #Pro Asn Val Lys Glu Ala
           610            - #     615            - #     620
           gcc ctc att gcc ctc cgg agc - #ctg caa cag gag cct ggc 4199
           Ala Leu Ile Ala Leu Arg Ser - #Leu Gln Gln Glu Pro Gly
                   625    - #             630    - #             635
           atc cat cag gta ctg gtg tcc - #ctg ggt gcc agt gag aaa 4238
           Ile His Gln Val Leu Val Ser - #Leu Gly Ala Ser Glu Lys
                       - #    640             - #    645
           cta tcc ttg ctc tct ctg ggg - #aat cag tca ctg cca cac 4277
           Leu Ser Leu Leu Ser Leu Gly - #Asn Gln Ser Leu Pro His
               650        - #         655        - #         660
           agc agt cct agg cct gcc tct - #gcc aaa cac tgc agg aaa 4316
           Ser Ser Pro Arg Pro Ala Ser - #Ala Lys His Cys Arg Lys
                       - #665                 - #670
           ctc att cac ctc ctg agg cca - #gcc cat agc atg t 4350
           Leu Ile His Leu Leu Arg Pro - #Ala His Ser Met
           675            - #     680            - #     685
           gattccagat tcctgcggtc cagcctccaa ctttgg - #ttgc cagctctttc 4400
           ttattctact acacaagccg ccaactcaac tgagag - #ctaa agagactaga 4450
           aaagagataa gctgccaact caactgagaa caagaa - #acta gaagagattt 4500
           atatataaag cttcttcctt ctcccagatg caggat - #gttt tcaaccagta 4550
           aattttattg ctgttggtgc cagagaagag tccttt - #cttc tctacatcca 4600
           ggggcctttt ctccaataat gtgcctttaa ctctag - #ggac ctgcctcacg 4650
           gaccttaggg aaaaacctca acctgaaaga tctctt - #cctt tctggagctc 4700
           ctttaatctt cccagcaggt ttttgcctta gacgtg - #ctgg ccccaggaca 4750
           gtgatgaaga cagagcctgt ctcagctcta ggctgt - #gggg atcaatgcca 4800
           tcagtccctg ttattgaggg attatccctt agccaa - #catt cctatctgtg 4850
           ggtgggcgtg gagagtgtat ctttttttgg ggtgtg - #tgtg tatatgtgtg 4900
           tgtgtatgtg tgtgtgtgtt taatagttct gtttgt - #aaac tcttttaata 4950
           aaagttgtgc ctcaccatac ttgaagctcc caggac - #aagg gttgagaggc 5000
           tcaacccctc tttcagcttc tatgtggtgt tggagg - #tgct ggtatcgtgt 5050
           tcacacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa - #aaaa aaaaaaaaaa 5100
           aaaaaaaaaa aaaaaaaaaa aaaaa 5125
      - -  - - <210> SEQ ID NO 5
     <211> LENGTH: 648
     <212> TYPE: PRT
     <213> ORGANISM: Homo sapiens
      - - <400> SEQUENCE: 5
      - -       Met Glu Lys Tyr His Val Leu - #Glu Met Ile Gly Glu Gly Ser
    Phe
              1          - #     5             - #     10             - #
    15
           Gly Arg Val Tyr Lys Gly Arg - #Arg Lys Tyr Ser Ala Gln Val Val
                       - #     20             - #     25             - #
    30
            Ala Leu Lys Phe Ile Pro Lys - #Leu Gly Arg Ser Glu Lys Glu Leu
                       - #     35             - #     40             - #
    45
            Arg Asn Leu Gln Arg Glu Ile - #Glu Ile Met Arg Gly Leu Arg His
                       - #     50             - #     55             - #
    60
            Pro Asn Ile Val His Met Leu - #Asp Ser Phe Glu Thr Asp Lys Glu
                       - #     65             - #     70             - #
    75
            Val Val Val Val Thr Asp Tyr - #Ala Glu Gly Glu Leu Phe Gln Ile
                       - #     80             - #     85             - #
    90
            Leu Glu Asp Asp Gly Lys Leu - #Pro Glu Asp Gln Val Gln Ala Ile
                       - #     95             - #    100             - #
    105
            Ala Ala Gln Leu Val Ser Ala - #Leu Tyr Tyr Leu His Ser His Arg
                       - #    110             - #    115             - #
    120
            Ile Leu His Arg Asp Met Lys - #Pro Gln Asn Ile Leu Leu Ala Lys
                       - #    125             - #    130             - #
    135
            Gly Gly Gly Ile Lys Leu Cys - #Asp Phe Gly Phe Ala Arg Ala Met
                       - #    140             - #    145             - #
    150
            Ser Thr Asn Thr Met Val Leu - #Thr Ser Ile Lys Gly Thr Pro Leu
                       - #    155             - #    160             - #
    165
            Tyr Met Ser Pro Glu Leu Val - #Glu Glu Arg Pro Tyr Asp His Thr
                       - #    170             - #    175             - #
    180
            Ala Asp Leu Trp Ser Val Gly - #Cys Ile Leu Tyr Glu Leu Ala Val
                       - #    185             - #    190             - #
    195
            Gly Thr Pro Pro Phe Tyr Ala - #Thr Ser Ile Phe Gln Leu Val Ser
                       - #    200             - #    205             - #
    210
            Leu Ile Leu Lys Asp Pro Val - #Arg Trp Pro Ser Thr Ile Ser Pro
                       - #    215             - #    220             - #
    225
            Cys Phe Lys Asn Phe Leu Gln - #Gly Leu Leu Thr Lys Asp Pro Arg
                       - #    230             - #    235             - #
    240
            Gln Arg Leu Ser Trp Pro Asp - #Leu Leu Tyr His Pro Phe Ile Ala
                       - #    245             - #    250             - #
    255
            Gly His Val Thr Ile Ile Thr - #Glu Pro Ala Gly Pro Asp Leu Gly
                       - #    260             - #    265             - #
    270
            Thr Pro Phe Thr Ser Arg Leu - #Pro Pro Glu Leu Gln Val Leu Lys
                       - #    275             - #    280             - #
    285
            Asp Glu Gln Ala His Arg Leu - #Ala Pro Lys Gly Asn Gln Ser Arg
                       - #    290             - #    295             - #



    300
            Ile Leu Thr Gln Ala Tyr Lys - #Arg Met Ala Glu Glu Ala Met Gln
                       - #    305             - #    310             - #
    315
            Lys Lys His Gln Asn Thr Gly - #Pro Ala Leu Glu Gln Glu Asp Lys
                       - #    320             - #    325             - #
    330
            Thr Ser Lys Val Ala Pro Gly - #Thr Ala Pro Leu Pro Arg Leu Gly
                       - #    335             - #    340             - #
    345
            Ala Thr Pro Gln Glu Ser Ser - #Leu Leu Ala Gly Ile Leu Ala Ser
                       - #    350             - #    355             - #
    360
            Glu Leu Lys Ser Ser Trp Ala - #Lys Ser Gly Thr Gly Glu Val Pro
                       - #    365             - #    370             - #
    375
            Ser Ala Pro Arg Glu Asn Arg - #Thr Thr Pro Asp Cys Glu Arg Ala
                       - #    380             - #    385             - #
    390
            Phe Pro Glu Glu Arg Pro Glu - #Val Leu Gly Gln Arg Ser Thr Asp
                       - #    395             - #    400             - #
    405
            Val Val Asp Leu Glu Asn Glu - #Glu Pro Asp Ser Asp Asn Glu Trp
                       - #    410             - #    415             - #
    420
            Gln His Leu Leu Glu Thr Thr - #Glu Pro Val Pro Ile Gln Leu Lys
                       - #    425             - #    430             - #
    435
            Ala Pro Leu Thr Leu Leu Cys - #Asn Pro Asp Phe Cys Gln Arg Ile
                       - #    440             - #    445             - #
    450
            Gln Ser Gln Leu His Glu Ala - #Gly Gly Gln Ile Leu Lys Gly Ile
                       - #    455             - #    460             - #
    465
            Leu Glu Gly Ala Ser His Ile - #Leu Pro Ala Phe Arg Val Leu Ser
                       - #    470             - #    475             - #
    480
            Ser Leu Leu Ser Ser Cys Ser - #Asp Ser Val Ala Leu Tyr Ser Phe
                       - #    485             - #    490             - #
    495
            Cys Arg Glu Ala Gly Leu Pro - #Gly Leu Leu Leu Ser Leu Leu Arg
                       - #    500             - #    505             - #
    510
            His Ser Gln Glu Ser Asn Ser - #Leu Gln Gln Gln Ser Trp Tyr Gly
                       - #    515             - #    520             - #
    525
            Thr Phe Leu Gln Asp Leu Met - #Ala Val Ile Gln Ala Tyr Phe Ala
                       - #    530             - #    535             - #
    540
            Cys Thr Phe Asn Leu Glu Arg - #Ser Gln Thr Ser Asp Ser Leu Gln
                       - #    545             - #    550             - #
    555
            Val Phe Gln Glu Ala Ala Asn - #Leu Phe Leu Asp Leu Leu Gly Lys
                       - #    560             - #    565             - #
    570
            Leu Leu Ala Gln Pro Asp Asp - #Ser Glu Gln Thr Leu Gln Arg Asp
                       - #    575             - #    580             - #
    585
            Ser Leu Met Cys Phe Thr Val - #Leu Cys Glu Ala Met Asp Gly Asn
                       - #    590             - #    595             - #
    600
            Ser Arg Ala Ile Ser Lys Ala - #Phe Tyr Ser Ser Leu Leu Thr Thr
                       - #    605             - #    610             - #
    615
            Gln Gln Val Val Leu Asp Gly - #Leu Leu His Gly Leu Thr Val Pro
                       - #    620             - #    625             - #
    630
            Gln Leu Pro Val His Thr Pro - #Gln Gly Asn Gln Ser Gly Glu Gly
                       - #    635             - #    640             - #
    645
            Arg Phe Ser
                    648
       - -  - - <210> SEQ ID NO 6
      <211> LENGTH: 5252
      <212> TYPE: DNA
      <213> ORGANISM: Homo sapiens
       - - <400> SEQUENCE: 6
       - -       ggagcttgga gctcctaggc tgggggcgtc ccagat - #gttg tggaactgtc
    50
            cctggatcta tagctcttca ccgtctctac tttctt - #cctt ctaagagatc 100
           ctgaaacctc tgtc atg gaa aag tac - #cac gtg ttg gag atg 141
                       - #    Met Glu Lys Tyr His Val Leu Glu - #Met
                       - #      1            - #   5
           att gga gaa ggc tct ttt ggg - #agg gtg tac aag ggt cga 180
           Ile Gly Glu Gly Ser Phe Gly - #Arg Val Tyr Lys Gly Arg
            10           - #       15           - #       20
           aga aaa tac agt gct cag gtc - #gtg gcc ctg aag ttc atc 219
           Arg Lys Tyr Ser Ala Gln Val - #Val Ala Leu Lys Phe Ile
                    25   - #               30   - #               35
           cca aaa ttg ggg cgc tca gag - #aag gag ctg agg aat ttg 258
           Pro Lys Leu Gly Arg Ser Glu - #Lys Glu Leu Arg Asn Leu
                       - #     40             - #     45
           caa cga gag att gaa ata atg - #cgg ggt ctg cgg cat ccc 297
           Gln Arg Glu Ile Glu Ile Met - #Arg Gly Leu Arg His Pro
                50       - #           55       - #           60
           aac att gtg cat atg ctt gac - #agc ttt gaa act gat aaa 336
           Asn Ile Val His Met Leu Asp - #Ser Phe Glu Thr Asp Lys
                       - # 65                 - # 70
           gag gtg gtg gtg gtg aca gac - #tat gct gag gga gag ctc 375
           Glu Val Val Val Val Thr Asp - #Tyr Ala Glu Gly Glu Leu
            75           - #       80           - #       85
           ttt cag atc cta gaa gat gac - #gga aaa ctt cct gaa gac 414
           Phe Gln Ile Leu Glu Asp Asp - #Gly Lys Leu Pro Glu Asp
                    90   - #               95   - #              100
           cag gtt cag gcc att gct gcc - #cag ttg gtg tca gcc ctg 453
           Gln Val Gln Ala Ile Ala Ala - #Gln Leu Val Ser Ala Leu
                       - #    105             - #    110
           tac tat ctg cat tcc cac cgc - #atc cta cac cga gat atg 492
           Tyr Tyr Leu His Ser His Arg - #Ile Leu His Arg Asp Met
               115        - #         120        - #         125
           aag cct cag aac atc ctc ctc - #gcc aag ggt ggt ggc atc 531
           Lys Pro Gln Asn Ile Leu Leu - #Ala Lys Gly Gly Gly Ile
                       - #130                 - #135
           aag ctc tgt gac ttt gga ttt - #gcc cgg gct atg agc acc 570
           Lys Leu Cys Asp Phe Gly Phe - #Ala Arg Ala Met Ser Thr
           140            - #     145            - #     150
           aat aca atg gtg ctg aca tcc - #atc aaa ggc aca cca ctc 609
           Asn Thr Met Val Leu Thr Ser - #Ile Lys Gly Thr Pro Leu
                   155    - #             160    - #             165
           tat atg tct cca gag ctg gtg - #gag gag cga cca tac gac 648
           Tyr Met Ser Pro Glu Leu Val - #Glu Glu Arg Pro Tyr Asp
                       - #    170             - #    175
           cac aca gcg gac ctc tgg tct - #gtt ggc tgc ata cta tat 687
           His Thr Ala Asp Leu Trp Ser - #Val Gly Cys Ile Leu Tyr
               180        - #         185        - #         190
           gaa ctg gca gta ggc acc cct - #ccc ttc tat gct aca agc 726
           Glu Leu Ala Val Gly Thr Pro - #Pro Phe Tyr Ala Thr Ser
                       - #195                 - #200
           atc ttt cag ctg gtc agc ctc - #att ctc aag gac cct gtg 765
           Ile Phe Gln Leu Val Ser Leu - #Ile Leu Lys Asp Pro Val
           205            - #     210            - #     215
           cgc tgg ccc tca acc atc agt - #ccc tgc ttt aag aac ttc 804
           Arg Trp Pro Ser Thr Ile Ser - #Pro Cys Phe Lys Asn Phe
                   220    - #             225    - #             230
           ctg cag gga ctg ctc acc aaa - #gac cca cgg cag cga ctg 843
           Leu Gln Gly Leu Leu Thr Lys - #Asp Pro Arg Gln Arg Leu
                       - #    235             - #    240
           tcc tgg cca gac ctc tta tat - #cac ccc ttt att gct ggt 882
           Ser Trp Pro Asp Leu Leu Tyr - #His Pro Phe Ile Ala Gly
               245        - #         250        - #         255
           cat gtc acc ata ata act gag - #cca gca ggc cca gat ttg 921
           His Val Thr Ile Ile Thr Glu - #Pro Ala Gly Pro Asp Leu
                       - #260                 - #265
           ggg acc cca ttc acc agc cgc - #cta ccc cca gaa ctt cag 960
           Gly Thr Pro Phe Thr Ser Arg - #Leu Pro Pro Glu Leu Gln
           270            - #     275            - #     280
           gtc cta aag gac gaa cag gcc - #cat cgg ttg gcc ccc aag 999
           Val Leu Lys Asp Glu Gln Ala - #His Arg Leu Ala Pro Lys
                   285    - #             290    - #             295
           ggt aat cag tct cgc atc ttg - #act cag gcc tat aaa cgc 1038
           Gly Asn Gln Ser Arg Ile Leu - #Thr Gln Ala Tyr Lys Arg
                       - #    300             - #    305
           atg gct gag gag gcc atg cag - #aag aaa cat cag aac aca 1077
           Met Ala Glu Glu Ala Met Gln - #Lys Lys His Gln Asn Thr
               310        - #         315        - #         320
           gga cct gcc ctt gag caa gag - #gac aag acc agc aag gtg 1116
           Gly Pro Ala Leu Glu Gln Glu - #Asp Lys Thr Ser Lys Val
                       - #325                 - #330
           gct cct ggc aca gcc cct ctg - #ccc aga ctc ggg gcc act 1155
           Ala Pro Gly Thr Ala Pro Leu - #Pro Arg Leu Gly Ala Thr
           335            - #     340            - #     345
           cct cag gaa tca agc ctc ctg - #gcc ggg atc tta gcc tca 1194
           Pro Gln Glu Ser Ser Leu Leu - #Ala Gly Ile Leu Ala Ser
                   350    - #             355    - #             360
           gaa ttg aag agc agc tgg gct - #aaa tca ggg act gga gag 1233
           Glu Leu Lys Ser Ser Trp Ala - #Lys Ser Gly Thr Gly Glu
                       - #    365             - #    370
           gtg ccc tct gca cct cgg gaa - #aac cgg acc acc cca gat 1272
           Val Pro Ser Ala Pro Arg Glu - #Asn Arg Thr Thr Pro Asp
               375        - #         380        - #         385
           tgt gaa cga gca ttc cca gag - #gag agg cca gag gtg ctg 1311
           Cys Glu Arg Ala Phe Pro Glu - #Glu Arg Pro Glu Val Leu
                       - #390                 - #395
           ggc cag cgg agc act gat gta - #gtg gac ctg gaa aat gag 1350
           Gly Gln Arg Ser Thr Asp Val - #Val Asp Leu Glu Asn Glu
           400            - #     405            - #     410
           gag cca gac agt gac aat gag - #tgg cag cac ctg cta gag 1389
           Glu Pro Asp Ser Asp Asn Glu - #Trp Gln His Leu Leu Glu
                   415    - #             420    - #             425
           acc act gag cct gtg cct att - #caa ctg aag gct cct ctc 1428
           Thr Thr Glu Pro Val Pro Ile - #Gln Leu Lys Ala Pro Leu
                       - #    430             - #    435
           acc ttg ctg tgt aat cct gac - #ttc tgc cag cgc atc cag 1467
           Thr Leu Leu Cys Asn Pro Asp - #Phe Cys Gln Arg Ile Gln
               440        - #         445        - #         450
           agt cag ctg cat gaa gct gga - #ggg cag atc ctg aaa ggc 1506
           Ser Gln Leu His Glu Ala Gly - #Gly Gln Ile Leu Lys Gly
                       - #455                 - #460
           atc ttg gag ggt gct tcc cac - #atc ctg cct gca ttc cgg 1545
           Ile Leu Glu Gly Ala Ser His - #Ile Leu Pro Ala Phe Arg
           465            - #     470            - #     475
           gtc ctg agc agt ctt ctc tcc - #agc tgc agt gat tct gtt 1584
           Val Leu Ser Ser Leu Leu Ser - #Ser Cys Ser Asp Ser Val
                   480    - #             485    - #             490
           gcc ttg tat tcc ttc tgc cgg - #gag gca ggg ctt cct ggg 1623
           Ala Leu Tyr Ser Phe Cys Arg - #Glu Ala Gly Leu Pro Gly
                       - #    495             - #    500
           ctg ctg ctg agt cta ctc agg - #cac agt cag gag agc aac 1662
           Leu Leu Leu Ser Leu Leu Arg - #His Ser Gln Glu Ser Asn
               505        - #         510        - #         515
           agc ctc cag cag caa tct tgg - #tat ggg acc ttc tta cag 1701
           Ser Leu Gln Gln Gln Ser Trp - #Tyr Gly Thr Phe Leu Gln
                       - #520                 - #525
           gac ctg atg gct gtg att cag - #gcc tac ttt gcc tgt acc 1740
           Asp Leu Met Ala Val Ile Gln - #Ala Tyr Phe Ala Cys Thr
           530            - #     535            - #     540
           ttc aat ctg gag agg agc cag - #aca agt gac agc ctg cag 1779
           Phe Asn Leu Glu Arg Ser Gln - #Thr Ser Asp Ser Leu Gln
                   545    - #             550    - #             555
           gtg ttt cag gag gct gcc aac - #ctt ttt ctg gac ctg ttg 1818
           Val Phe Gln Glu Ala Ala Asn - #Leu Phe Leu Asp Leu Leu
                       - #    560             - #    565
           ggg aaa ctg ctg gcc caa cca - #gat gac tct gag cag act 1857
           Gly Lys Leu Leu Ala Gln Pro - #Asp Asp Ser Glu Gln Thr
               570        - #         575        - #         580
           ttg cgg agg gac agc ctt atg - #tgc ttt act gtc ctg tgc 1896
           Leu Arg Arg Asp Ser Leu Met - #Cys Phe Thr Val Leu Cys
                       - #585                 - #590
           gaa gcc atg gat ggg aac agc - #cgg gcc atc tcc aaa gcc 1935
           Glu Ala Met Asp Gly Asn Ser - #Arg Ala Ile Ser Lys Ala
           595            - #     600            - #     605
           ttt tac tcc agc ttg ctg acg - #aca cag cag gtt gtc ttg 1974
           Phe Tyr Ser Ser Leu Leu Thr - #Thr Gln Gln Val Val Leu
                   610    - #             615    - #             620
           gat ggg ctc ctt cat ggc ttg - #aca gtt cca cag ctc cct 2013
           Asp Gly Leu Leu His Gly Leu - #Thr Val Pro Gln Leu Pro
                       - #    625             - #    630
           gtc cac act ccc caa ggt tcc - #cta ctc ctg ctg cca tgt 2052
           Val His Thr Pro Gln Gly Ser - #Leu Leu Leu Leu Pro Cys
               635        - #         640        - #         645
           cgg tga g t actggtgcta ttgtctaggg - # caagagcctc 2090
           Arg Xaa
               648
           aggcctttgg  agt tac tct ttg ct - #t ttc tcc aca gga gcc 2130
                       - #Ser Tyr Ser Leu Leu Phe Ser Thr Gly Ala
                       - #  1               5 - #                 10
           ccg caa gtg agc cag cca ctg - #cga gag cag agt gag gat 2169
           Pro Gln Val Ser Gln Pro Leu - #Arg Glu Gln Ser Glu Asp
                       - #     15             - #     20
           ata cct gga gcc att tcc tct - #gcc ctg gca gcc ata tgc 2208
           Ile Pro Gly Ala Ile Ser Ser - #Ala Leu Ala Ala Ile Cys
                25       - #           30       - #           35
           act gct cct gtg gga ctg ccc - #gac tgc tgg gat gcc aag 2247
           Thr Ala Pro Val Gly Leu Pro - #Asp Cys Trp Asp Ala Lys
                       - # 40                 - # 45
           gag cag gtc tgt tgg cat ttg - #gca aat cag cta act gaa 2286
           Glu Gln Val Cys Trp His Leu - #Ala Asn Gln Leu Thr Glu
            50           - #       55           - #       60
           gac agc agc cag ctc agg cca - #tcc ctc atc tct ggc ctg 2325
           Asp Ser Ser Gln Leu Arg Pro - #Ser Leu Ile Ser Gly Leu
                    65   - #               70   - #               75
           cag cat ccc atc ctg tgc ctg - #cac ctt ctc aag gtt cta 2364
           Gln His Pro Ile Leu Cys Leu - #His Leu Leu Lys Val Leu
                       - #     80             - #     85
           tac tcc tgc tgc ctt gtc agt - #gag ggc ctg tgc cgt ctt 2403



           Tyr Ser Cys Cys Leu Val Ser - #Glu Gly Leu Cys Arg Leu
                90       - #           95       - #          100
           ctg ggg cag gag ccc ctg gcc - #ttg gaa tcc ctg ttt atg 2442
           Leu Gly Gln Glu Pro Leu Ala - #Leu Glu Ser Leu Phe Met
                       - #105                 - #110
           ttg att cag ggc aag gta aaa - #gta gta gat tgg gaa gag 2481
           Leu Ile Gln Gly Lys Val Lys - #Val Val Asp Trp Glu Glu
           115            - #     120            - #     125
           tct act gaa gtg aca ctc tac - #ttc ctc tcc ctt ctt gtc 2520
           Ser Thr Glu Val Thr Leu Tyr - #Phe Leu Ser Leu Leu Val
                   130    - #             135    - #             140
           ttt cgg ctc caa aac ctg cct - #tgt gga atg gag aag cta 2559
           Phe Arg Leu Gln Asn Leu Pro - #Cys Gly Met Glu Lys Leu
                       - #    145             - #    150
           ggc agt gac gtt gct act ctc - #ttt acc cat tcg cat gtc 2598
           Gly Ser Asp Val Ala Thr Leu - #Phe Thr His Ser His Val
               155        - #         160        - #         165
           gtc tct ctt gtg agt gca gca - #gcc tgt cta ttg gga cag 2637
           Val Ser Leu Val Ser Ala Ala - #Ala Cys Leu Leu Gly Gln
                       - #170                 - #175
           ctt ggt cag caa ggg gtg acc - #ttt gac ctc cag ccc atg 2676
           Leu Gly Gln Gln Gly Val Thr - #Phe Asp Leu Gln Pro Met
           180            - #     185            - #     190
           gaa tgg atg gct gca gcc aca - #cat gcc ttg tct gcc cct 2715
           Glu Trp Met Ala Ala Ala Thr - #His Ala Leu Ser Ala Pro
                   195    - #             200    - #             205
           gca gag ctc ctc act gag gta - #cag atg gat ctt ggg atg 2754
           Ala Glu Leu Leu Thr Glu Val - #Gln Met Asp Leu Gly Met
                       - #    210             - #    215
           gat ggg aag taaagag agaggaactg gg - #cattttgg ggagcctctg 2800
           Asp Gly Lys
               220 221
           gaccagagga atgaagaagc aacccacagc cttccc - #tctc aagctactgt 2850
           gcctgtgata gccttggaac ttccccgcct gccctc - #agta ctgacccttt 2900
           gaaggaaacc attcgctgcg tcccctggga tccagt - #ggga gataaaatga 2950
           attccctggg tttcagcaga catacacatg agttgt - #gagg tcagagggtt 3000
           aaggtttgat aagaaaatga aataagacga caggga - #aata ctaggtggga 3050
           aagcggaagg aattatttct gggacttcct ttactt - #gtaa gtcagggaca 3100
           ggaatgaata aaagcatttg gattcctgac ttctgt - #cttt ccccccgccc 3150
           tctttcactt ttatctctag caggggaagg ctagcc - #taat cagggatatg 3200
           tccagttcag aaatgtggac cgttttgtgg caccgc - #ttct ccatggtcct 3250
           gaggctcccc gaggaggcat ctgcacagga agggga - #gctt tcgctatcca 3300
           gtccaccaag ccctgagcca gactggacac tgattt - #ctcc ccagggcatg 3350
           gcagccctgc tgagcctggc catggccacc tttacc - #cagg agccccagtt 3400
           atgcctgagc tgcctgtccc agcatggaag tatcct - #catg tccatcctga 3450
           agcatctgct ttgccccagc ttcctgaatc aactgc - #gcca ggcgcctcat 3500
           gggtctgagt ttctccctgt cgtggtgctc tctgtc - #tgcc agctcctttg 3550
           cttccccttt gcgctggaca tggatgctga cctcct - #tata ggtgtcttgg 3600
           ccgacctcag ggactcagaa gttgcagccc atctgc - #tgca ggtctgctgc 3650
           taccatcttc cgttgatgca agtggagctg cccatc - #agcc ttctcacacg 3700
           cctggccctc atggatccca cctctctcaa ccagtt - #tgtg aacacagtgt 3750
           ctgcctcccc tagaaccatc gtctcgtttc tctcag - #ttgc cctcctgagt 3800
           gaccagccac tgttgacctc cgaccttctc tctctg - #ctgg cccatactgc 3850
           cagggtcctg tctcccagcc acttgtcctt tatcca - #agag cttctggctg 3900
           gctctgatga atcctatcgg cccctgcgca gcctcc - #tggg ccacccagag 3950
           aattctgtgc gggcacacac ttataggctc ctggga - #cact tgctccaaca 4000
           cagcatggcc ctgcgtgggg cactgcagag ccagtc - #tgga ctgctcagcc 4050
           ttctgctgct tgggcttgga gacaaggatc ctgttg - #tgcg gtgcagtgcc 4100
           agctttgctg tgggcaatgc agcctaccag gctggt - #cctc tgggacctgc 4150
           cctggcagct gcagtgccca gtatgaccca gctgct - #tgga gatcctcagg 4200
           ctggtatccg gcgcaatgtt gcatcagctc tgggca - #actt gggacctgaa 4250
           ggtttgggag aggagctgtt acagtgcgaa gtaccc - #cagc ggctcctaga 4300
           aatggcatgt ggagaccccc agccaaatgt gaagga - #ggct gccctcattg 4350
           ccctccggag cctgcaacag gagcctggca tccatc - #aggt actggtgtcc 4400
           ctgggtgcca gtgagaaact atccttgctc tctctg - #ggga atcagtcact 4450
           gccacacagc agtcctaggc ctgcctctgc caaaca - #ctgc aggaaactca 4500
           ttcacctcct gaggccagcc catagcatgt gattcc - #agat tcctgcggtc 4550
           cagcctccaa ctttggttgc cagctctttc ttattc - #tact acacaagccg 4600
           ccaactcaac tgagagctaa agagactaga aaagag - #ataa gctgccaact 4650
           caactgagaa caagaaacta gaagagattt atatat - #aaag cttcttcctt 4700
           ctcccagatg caggatgttt tcaaccagta aatttt - #attg ctgttggtgc 4750
           cagagaagag tcctttcttc tctacatcca ggggcc - #tttt ctccaataat 4800
           gtgcctttaa ctctagggac ctgcctcacg gacctt - #aggg aaaaacctca 4850
           acctgaaaga tctcttcctt tctggagctc ctttaa - #tctt cccagcaggt 4900
           ttttgcctta gacgtgctgg ccccaggaca gtgatg - #aaga cagagcctgt 4950
           ctcagctcta ggctgtgggg atcaatgcca tcagtc - #cctg ttattgaggg 5000
           attatccctt agccaacatt cctatctgtg ggtggg - #cgtg gagagtgtat 5050
           ctttttttgg ggtgtgtgtg tatatgtgtg tgtgta - #tgtg tgtgtgtgtt 5100
           taatagttct gtttgtaaac tcttttaata aaagtt - #gtgc ctcaccatac 5150
           ttgaagctcc caggacaagg gttgagaggc tcaacc - #cctc tttcagcttc 5200
           tatgtggtgt tggaggtgct ggtatcgtgt tcacac - #aaaa aaaaaaaaaa 5250
           aa 5252
      - -  - - <210> SEQ ID NO 7
     <211> LENGTH: 647
     <212> TYPE: PRT
     <213> ORGANISM: Homo sapiens
      - - <400> SEQUENCE: 7
      - -       Met Glu Lys Tyr His Val Leu - #Glu Met Ile Gly Glu Gly Ser
    Phe
              1          - #     5             - #     10             - #
    15
           Gly Arg Val Tyr Lys Gly Arg - #Arg Lys Tyr Ser Ala Gln Val Val
                       - #     20             - #     25             - #
    30
            Ala Leu Lys Phe Ile Pro Lys - #Leu Gly Arg Ser Glu Lys Glu Leu
                       - #     35             - #     40             - #
    45
            Arg Asn Leu Gln Arg Glu Ile - #Glu Ile Met Arg Gly Leu Arg His
                       - #     50             - #     55             - #
    60
            Pro Asn Ile Val His Met Leu - #Asp Ser Phe Glu Thr Asp Lys Glu
                       - #     65             - #     70             - #
    75
            Val Val Val Val Thr Asp Tyr - #Ala Glu Gly Glu Leu Phe Gln Ile
                       - #     80             - #     85             - #
    90
            Leu Glu Asp Asp Gly Lys Leu - #Pro Glu Asp Gln Val Gln Ala Ile
                       - #     95             - #    100             - #
    105
            Ala Ala Gln Leu Val Ser Ala - #Leu Tyr Tyr Leu His Ser His Arg
                       - #    110             - #    115             - #
    120
            Ile Leu His Arg Asp Met Lys - #Pro Gln Asn Ile Leu Leu Ala Lys
                       - #    125             - #    130             - #
    135
            Gly Gly Gly Ile Lys Leu Cys - #Asp Phe Gly Phe Ala Arg Ala Met
                       - #    140             - #    145             - #
    150
            Ser Thr Asn Thr Met Val Leu - #Thr Ser Ile Lys Gly Thr Pro Leu
                       - #    155             - #    160             - #
    165
            Tyr Met Ser Pro Glu Leu Val - #Glu Glu Arg Pro Tyr Asp His Thr
                       - #    170             - #    175             - #
    180
            Ala Asp Leu Trp Ser Val Gly - #Cys Ile Leu Tyr Glu Leu Ala Val
                       - #    185             - #    190             - #
    195
            Gly Thr Pro Pro Phe Tyr Ala - #Thr Ser Ile Phe Gln Leu Val Ser
                       - #    200             - #    205             - #
    210
            Leu Ile Leu Lys Asp Pro Val - #Arg Trp Pro Ser Thr Ile Ser Pro
                       - #    215             - #    220             - #
    225
            Cys Phe Lys Asn Phe Leu Gln - #Gly Leu Leu Thr Lys Asp Pro Arg
                       - #    230             - #    235             - #
    240
            Gln Arg Leu Ser Trp Pro Asp - #Leu Leu Tyr His Pro Phe Ile Ala
                       - #    245             - #    250             - #
    255
            Gly His Val Thr Ile Ile Thr - #Glu Pro Ala Gly Pro Asp Leu Gly
                       - #    260             - #    265             - #
    270
            Thr Pro Phe Thr Ser Arg Leu - #Pro Pro Glu Leu Gln Val Leu Lys
                       - #    275             - #    280             - #
    285
            Asp Glu Gln Ala His Arg Leu - #Ala Pro Lys Gly Asn Gln Ser Arg
                       - #    290             - #    295             - #
    300
            Ile Leu Thr Gln Ala Tyr Lys - #Arg Met Ala Glu Glu Ala Met Gln
                       - #    305             - #    310             - #
    315
            Lys Lys His Gln Asn Thr Gly - #Pro Ala Leu Glu Gln Glu Asp Lys
                       - #    320             - #    325             - #
    330
            Thr Ser Lys Val Ala Pro Gly - #Thr Ala Pro Leu Pro Arg Leu Gly
                       - #    335             - #    340             - #
    345
            Ala Thr Pro Gln Glu Ser Ser - #Leu Leu Ala Gly Ile Leu Ala Ser
                       - #    350             - #    355             - #
    360
            Glu Leu Lys Ser Ser Trp Ala - #Lys Ser Gly Thr Gly Glu Val Pro
                       - #    365             - #    370             - #
    375
            Ser Ala Pro Arg Glu Asn Arg - #Thr Thr Pro Asp Cys Glu Arg Ala
                       - #    380             - #    385             - #
    390
            Phe Pro Glu Glu Arg Pro Glu - #Val Leu Gly Gln Arg Ser Thr Asp
                       - #    395             - #    400             - #
    405
            Val Val Asp Leu Glu Asn Glu - #Glu Pro Asp Ser Asp Asn Glu Trp
                       - #    410             - #    415             - #
    420
            Gln His Leu Leu Glu Thr Thr - #Glu Pro Val Pro Ile Gln Leu Lys
                       - #    425             - #    430             - #
    435
            Ala Pro Leu Thr Leu Leu Cys - #Asn Pro Asp Phe Cys Gln Arg Ile
                       - #    440             - #    445             - #
    450
            Gln Ser Gln Leu His Glu Ala - #Gly Gly Gln Ile Leu Lys Gly Ile
                       - #    455             - #    460             - #
    465
            Leu Glu Gly Ala Ser His Ile - #Leu Pro Ala Phe Arg Val Leu Ser
                       - #    470             - #    475             - #
    480
            Ser Leu Leu Ser Ser Cys Ser - #Asp Ser Val Ala Leu Tyr Ser Phe
                       - #    485             - #    490             - #
    495
            Cys Arg Glu Ala Gly Leu Pro - #Gly Leu Leu Leu Ser Leu Leu Arg
                       - #    500             - #    505             - #
    510
            His Ser Gln Glu Ser Asn Ser - #Leu Gln Gln Gln Ser Trp Tyr Gly
                       - #    515             - #    520             - #
    525
            Thr Phe Leu Gln Asp Leu Met - #Ala Val Ile Gln Ala Tyr Phe Ala
                       - #    530             - #    535             - #
    540
            Cys Thr Phe Asn Leu Glu Arg - #Ser Gln Thr Ser Asp Ser Leu Gln
                       - #    545             - #    550             - #
    555
            Val Phe Gln Glu Ala Ala Asn - #Leu Phe Leu Asp Leu Leu Gly Lys
                       - #    560             - #    565             - #
    570
            Leu Leu Ala Gln Pro Asp Asp - #Ser Glu Gln Thr Leu Arg Arg Asp
                       - #    575             - #    580             - #
    585
            Ser Leu Met Cys Phe Thr Val - #Leu Cys Glu Ala Met Asp Gly Asn
                       - #    590             - #    595             - #
    600
            Ser Arg Ala Ile Ser Lys Ala - #Phe Tyr Ser Ser Leu Leu Thr Thr
                       - #    605             - #    610             - #
    615
            Gln Gln Val Val Leu Asp Gly - #Leu Leu His Gly Leu Thr Val Pro
                       - #    620             - #    625             - #
    630
            Gln Leu Pro Val His Thr Pro - #Gln Gly Ser Leu Leu Leu Leu Pro
                       - #    635             - #    640             - #
    645
            Cys Arg
                647
       - -  - - <210> SEQ ID NO 8
      <211> LENGTH: 32
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 8
       - -       caatacaatg gtgctgacat ccatcaaagg ca - #32
       - -  - - <210> SEQ ID NO 9
      <211> LENGTH: 23
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 9
       - -       gaagggaggg gtgcctactg cca 23
       - -  - - <210> SEQ ID NO 10
      <211> LENGTH: 38
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 10
       - -       ctccagctct ggagacatat agagtggtgt gccttt - #ga 38
       - -  - - <210> SEQ ID NO 11
      <211> LENGTH: 60
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 11
       - -       ccatcgatgt acccatacga cgtcccagac tacgct - #gaaa agtaccacgt
    50
            gttggagatg 60
       - -  - - <210> SEQ ID NO 12
      <211> LENGTH: 32



      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 12
       - -       gctctagact aaggggcagg tcctgtgttc tg - #32
       - -  - - <210> SEQ ID NO 13
      <211> LENGTH: 21
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 13
       - -       ctgacgacac agcaggttgt c 21
       - -  - - <210> SEQ ID NO 14
      <211> LENGTH: 21
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 14
       - -       cagatgcttc aggatggaca t 21
       - -  - - <210> SEQ ID NO 15
      <211> LENGTH: 19
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 15
       - -       agagtagcaa cgtcactgc 19
       - -  - - <210> SEQ ID NO 16
      <211> LENGTH: 21
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 16
       - -       cctcactgac aaggcagcag g 21
       - -  - - <210> SEQ ID NO 17
      <211> LENGTH: 21
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 17
       - -       cccgaggagg catctgcaca g 21
       - -  - - <210> SEQ ID NO 18
      <211> LENGTH: 21
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 18
       - -       cagaacttca ggtcctaaag g 21
       - -  - - <210> SEQ ID NO 19
      <211> LENGTH: 32
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 19
       - -       tcgacaagca gggaacaccc aagtagaagc tc - #32
       - -  - - <210> SEQ ID NO 20
      <211> LENGTH: 32
      <212> TYPE: DNA
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 20
       - -       tcgacaagca gggaagtggg aagtagaagc tc - #32
       - -  - - <210> SEQ ID NO 21
      <211> LENGTH: 685
      <212> TYPE: PRT
      <213> ORGANISM: Homo sapiens
       - - <400> SEQUENCE: 21
       - -       Ser Tyr Ser Leu Leu Phe Ser - #Thr Gly Ala Pro Gln Val Ser
    Gln
              1          - #     5             - #     10             - #
    15
           Pro Leu Arg Glu Gln Ser Glu - #Asp Ile Pro Gly Ala Ile Ser Ser
                       - #     20             - #     25             - #
    30
            Ala Leu Ala Ala Ile Cys Thr - #Ala Pro Val Gly Leu Pro Asp Cys
                       - #     35             - #     40             - #
    45
            Trp Asp Ala Lys Glu Gln Val - #Cys Trp His Leu Ala Asn Gln Leu
                       - #     50             - #     55             - #
    60
            Thr Glu Asp Ser Ser Gln Leu - #Arg Pro Ser Leu Ile Ser Gly Leu
                       - #     65             - #     70             - #
    75
            Gln His Pro Ile Leu Cys Leu - #His Leu Leu Lys Val Leu Tyr Ser
                       - #     80             - #     85             - #
    90
            Cys Cys Leu Val Ser Glu Gly - #Leu Cys Arg Leu Leu Gly Gln Glu
                       - #     95             - #    100             - #
    105
            Pro Leu Ala Leu Glu Ser Leu - #Phe Met Leu Ile Gln Gly Lys Val
                       - #    110             - #    115             - #
    120
            Lys Val Val Asp Trp Glu Glu - #Ser Thr Glu Val Thr Leu Tyr Phe
                       - #    125             - #    130             - #
    135
            Leu Ser Leu Leu Val Phe Arg - #Leu Gln Asn Leu Pro Cys Gly Met
                       - #    140             - #    145             - #
    150
            Glu Lys Leu Gly Ser Asp Val - #Ala Thr Leu Phe Thr His Ser His
                       - #    155             - #    160             - #
    165
            Val Val Ser Leu Val Ser Ala - #Ala Ala Cys Leu Leu Gly Gln Leu
                       - #    170             - #    175             - #
    180
            Gly Gln Gln Gly Val Thr Phe - #Asp Leu Gln Pro Met Glu Trp Met
                       - #    185             - #    190             - #
    195
            Ala Ala Ala Thr His Ala Leu - #Ser Ala Pro Ala Glu Val Arg Leu
                       - #    200             - #    205             - #
    210
            Thr Pro Pro Gly Ser Cys Gly - #Phe Tyr Asp Gly Leu Leu Ile Leu
                       - #    215             - #    220             - #
    225
            Leu Leu Gln Leu Leu Thr Glu - #Gln Gly Lys Ala Ser Leu Ile Arg
                       - #    230             - #    235             - #
    240
            Asp Met Ser Ser Ser Glu Met - #Trp Thr Val Leu Trp His Arg Phe
                       - #    245             - #    250             - #
    255
            Ser Met Val Leu Arg Leu Pro - #Glu Glu Ala Ser Ala Gln Glu Gly
                       - #    260             - #    265             - #
    270
            Glu Leu Ser Leu Ser Ser Pro - #Pro Ser Pro Glu Pro Asp Trp Thr
                       - #    275             - #    280             - #
    285
            Leu Ile Ser Pro Gln Gly Met - #Ala Ala Leu Leu Ser Leu Ala Met
                       - #    290             - #    295             - #
    300
            Ala Thr Phe Thr Gln Glu Pro - #Gln Leu Cys Leu Ser Cys Leu Ser
                       - #    305             - #    310             - #
    315
            Gln His Gly Ser Ile Leu Met - #Ser Ile Leu Lys His Leu Leu Cys
                       - #    320             - #    325             - #
    330
            Pro Ser Phe Leu Asn Gln Leu - #Arg Gln Ala Pro His Gly Ser Glu
                       - #    335             - #    340             - #
    345
            Phe Leu Pro Val Val Val Leu - #Ser Val Cys Gln Leu Leu Cys Phe
                       - #    350             - #    355             - #
    360
            Pro Phe Ala Leu Asp Met Asp - #Ala Asp Leu Leu Ile Val Val Leu
                       - #    365             - #    370             - #
    375
            Ala Asp Leu Arg Asp Ser Glu - #Val Ala Ala His Leu Leu Gln Val
                       - #    380             - #    385             - #
    390
            Cys Cys Tyr His Leu Pro Leu - #Met Gln Val Glu Leu Pro Ile Ser
                       - #    395             - #    400             - #
    405
            Leu Leu Thr Arg Leu Ala Leu - #Met Asp Pro Thr Ser Leu Asn Gln
                       - #    410             - #    415             - #
    420
            Phe Val Asn Thr Val Ser Ala - #Ser Pro Arg Thr Ile Val Ser Phe
                       - #    425             - #    430             - #
    435
            Leu Ser Val Ala Leu Leu Ser - #Asp Gln Pro Leu Leu Thr Ser Asp
                       - #    440             - #    445             - #
    450
            Leu Leu Ser Leu Leu Ala His - #Thr Ala Arg Val Leu Ser Pro Ser
                       - #    455             - #    460             - #
    465
            His Leu Ser Phe Ile Gln Glu - #Leu Leu Ala Gly Ser Asp Glu Ser
                       - #    470             - #    475             - #
    480
            Tyr Arg Pro Leu Arg Ser Leu - #Leu Gly His Pro Glu Asn Ser Val
                       - #    485             - #    490             - #
    495
            Arg Ala His Thr Tyr Arg Leu - #Leu Gly His Leu Leu Gln His Ser
                       - #    500             - #    505             - #
    510
            Met Ala Leu Arg Gly Ala Leu - #Gln Ser Gln Ser Gly Leu Leu Ser
                       - #    515             - #    520             - #
    525
            Leu Leu Leu Leu Gly Leu Gly - #Asp Lys Asp Pro Val Val Arg Cys
                       - #    530             - #    535             - #
    540
            Ser Ala Ser Phe Ala Val Gly - #Asn Ala Ala Tyr Gln Ala Gly Pro
                       - #    545             - #    550             - #
    555
            Leu Gly Pro Ala Leu Ala Ala - #Ala Val Pro Ser Met Thr Gln Leu
                       - #    560             - #    565             - #
    570
            Leu Gly Asp Pro Gln Ala Gly - #Ile Arg Arg Asn Val Ala Ser Ala
                       - #    575             - #    580             - #
    585
            Leu Gly Asn Leu Gly Pro Glu - #Gly Leu Gly Glu Glu Leu Leu Gln
                       - #    590             - #    595             - #
    600
            Cys Glu Val Pro Gln Arg Leu - #Leu Glu Met Ala Cys Gly Asp Pro
                       - #    605             - #    610             - #
    615
            Gln Pro Asn Val Lys Glu Ala - #Ala Leu Ile Ala Leu Arg Ser Leu
                       - #    620             - #    625             - #
    630
            Gln Gln Glu Pro Gly Ile His - #Gln Val Leu Val Ser Leu Gly Ala
                       - #    635             - #    640             - #
    645
            Ser Glu Lys Leu Ser Leu Leu - #Ser Leu Gly Asn Gln Ser Leu Pro
                       - #    650             - #    655             - #
    660
            His Ser Ser Pro Arg Pro Ala - #Ser Ala Lys His Cys Arg Lys Leu
                       - #    665             - #    670             - #
    675
            Ile His Leu Leu Arg Pro Ala - #His Ser Met
                        - #    680             - #    685
       - -  - - <210> SEQ ID NO 22
      <211> LENGTH: 221
      <212> TYPE: PRT
      <213> ORGANISM: Homo sapiens
       - - <400> SEQUENCE: 22
       - -       Ser Tyr Ser Leu Leu Phe Ser - #Thr Gly Ala Pro Gln Val Ser
    Gln
              1          - #     5             - #     10             - #
    15
           Pro Leu Arg Glu Gln Ser Glu - #Asp Ile Pro Gly Ala Ile Ser Ser
                       - #     20             - #     25             - #
    30
            Ala Leu Ala Ala Ile Cys Thr - #Ala Pro Val Gly Leu Pro Asp Cys
                       - #     35             - #     40             - #
    45
            Trp Asp Ala Lys Glu Gln Val - #Cys Trp His Leu Ala Asn Gln Leu
                       - #     50             - #     55             - #
    60
            Thr Glu Asp Ser Ser Gln Leu - #Arg Pro Ser Leu Ile Ser Gly Leu
                       - #     65             - #     70             - #
    75
            Gln His Pro Ile Leu Cys Leu - #His Leu Leu Lys Val Leu Tyr Ser
                       - #     80             - #     85             - #
    90
            Cys Cys Leu Val Ser Glu Gly - #Leu Cys Arg Leu Leu Gly Gln Glu
                       - #     95             - #    100             - #
    105
            Pro Leu Ala Leu Glu Ser Leu - #Phe Met Leu Ile Gln Gly Lys Val
                       - #    110             - #    115             - #
    120
            Lys Val Val Asp Trp Glu Glu - #Ser Thr Glu Val Thr Leu Tyr Phe
                       - #    125             - #    130             - #
    135
            Leu Ser Leu Leu Val Phe Arg - #Leu Gln Asn Leu Pro Cys Gly Met
                       - #    140             - #    145             - #
    150
            Glu Lys Leu Gly Ser Asp Val - #Ala Thr Leu Phe Thr His Ser His
                       - #    155             - #    160             - #
    165
            Val Val Ser Leu Val Ser Ala - #Ala Ala Cys Leu Leu Gly Gln Leu
                       - #    170             - #    175             - #
    180



            Gly Gln Gln Gly Val Thr Phe - #Asp Leu Gln Pro Met Glu Trp Met
                       - #    185             - #    190             - #
    195
            Ala Ala Ala Thr His Ala Leu - #Ser Ala Pro Ala Glu Leu Leu Thr
                       - #    200             - #    205             - #
    210
            Glu Val Gln Met Asp Leu Gly - #Met Asp Gly Lys
                        - #    215             - #    220 221
       - -  - - <210> SEQ ID NO 23
      <211> LENGTH: 795
      <212> TYPE: PRT
      <213> ORGANISM: Drosophila virilis
       - - <400> SEQUENCE: 23
       - -       Met Asp Arg Tyr Ala Val Ser - #Ser Leu Val Gly Gln Gly Ser
    Phe
              1          - #     5             - #     10             - #
    15
           Gly Cys Val Tyr Lys Ala Gln - #Arg Arg Asp Asp Asp Lys Val Val
                       - #     20             - #     25             - #
    30
            Ala Ile Lys Val Ile Ser Lys - #Arg Gly Arg Ser Asn Arg Glu Leu
                       - #     35             - #     40             - #
    45
            Lys Asn Leu Arg Arg Glu Cys - #Asp Ile Gln Ala Arg Leu Lys His
                       - #     50             - #     55             - #
    60
            Pro His Val Ile Glu Met Val - #Glu Ser Phe Glu Ser Lys Phe Asp
                       - #     65             - #     70             - #
    75
            Leu Phe Val Val Thr Glu Phe - #Ala Leu Met Asp Leu His Arg Tyr
                       - #     80             - #     85             - #
    90
            Leu Ser Phe Asn Gly Ala Met - #Pro Glu Glu His Ala Gln Arg Val
                       - #     95             - #    100             - #
    105
            Val Cys His Leu Val Ser Ala - #Leu Tyr Tyr Leu His Ser Asn Arg
                       - #    110             - #    115             - #
    120
            Ile Leu His Arg Asp Leu Lys - #Pro Gln Asn Val Leu Leu Asp Lys
                       - #    125             - #    130             - #
    135
            Asn Met His Ala Lys Leu Cys - #Asp Phe Gly Leu Ala Arg Asn Met
                       - #    140             - #    145             - #
    150
            Thr Met Gly Thr His Val Leu - #Thr Ser Ile Lys Gly Thr Pro Leu
                       - #    155             - #    160             - #
    165
            Tyr Met Ala Pro Glu Leu Leu - #Ala Glu Gln Pro Tyr Asp His Gln
                       - #    170             - #    175             - #
    180
            Ala Asp Met Trp Ser Leu Gly - #Cys Ile Ala Tyr Glu Ser Met Ala
                       - #    185             - #    190             - #
    195
            Gly Gln Pro Pro Phe Cys Ala - #Thr Ser Ile Leu His Leu Val Lys
                       - #    200             - #    205             - #
    210
            Leu Ile Lys His Glu Asp Val - #Lys Trp Pro Ser Thr Leu Ser Ser
                       - #    215             - #    220             - #
    225
            Glu Cys Arg Ser Phe Leu Gln - #Gly Leu Leu Glu Lys Asp Pro Ser
                       - #    230             - #    235             - #
    240
            Met Arg Ile Ser Trp Thr Gln - #Leu Leu Cys His Pro Phe Val Glu
                       - #    245             - #    250             - #
    255
            Gly Lys Leu Tyr Ile Ala Glu - #Val Gln Ala Ala Gln Thr Ser Pro
                       - #    260             - #    265             - #
    270
            Phe Ile Asn Pro Gln Leu Ala - #Lys Asp Thr Lys Lys Ser Gln Gln
                       - #    275             - #    280             - #
    285
            Leu Arg His Val Gly Ala Asp - #Leu Gly Asp Val Leu Ala Ala Leu
                       - #    290             - #    295             - #
    300
            Lys Leu Ser Asp Val Ala Asn - #Glu Asn Leu Ser Thr Ser Arg Asp
                       - #    305             - #    310             - #
    315
            Ser Ile Asn Ala Ile Ala Pro - #Ser Asp Ile Glu Gln Leu Glu Thr
                       - #    320             - #    325             - #
    330
            Asp Val Glu Asp Asn Val His - #Arg Leu Ile Val Pro Phe Ala Asp
                       - #    335             - #    340             - #
    345
            Ile Ser Tyr Arg Glu Leu Pro - #Cys Gly Thr Ala Ala Ala Ala Arg
                       - #    350             - #    355             - #
    360
            Arg Ala Gly Ala Met Pro Leu - #Ile Asn Ser Gln Thr Cys Phe Val
                       - #    365             - #    370             - #
    375
            Ser Gly Asn Ser Asn Met Ile - #Leu Asn His Leu Asn Asp Asn Phe
                       - #    380             - #    385             - #
    390
            Ala Ile Glu Ala Pro Ala Ser - #Ser Ala Thr Lys Ser Met Lys Ser
                       - #    395             - #    400             - #
    405
            Lys Leu Lys Leu Ala Leu Asn - #Ile Lys Gln Ser Arg Ser Lys Asp
                       - #    410             - #    415             - #
    420
            Leu Glu Lys Arg Lys Leu Ser - #Gln Asn Leu Asp Asn Phe Ser Leu
                       - #    425             - #    430             - #
    435
            Arg Leu Gly Gln Ser Ile Asp - #Ile Glu Val Gln Arg Lys Thr Thr
                       - #    440             - #    445             - #
    450
            Glu Met Leu Thr Gln Gln Ser - #Gln Ala Gln Gln Leu Gln Asp Arg
                       - #    455             - #    460             - #
    465
            Lys Thr Gln Gln Leu Lys Gln - #Ser Met His Ser Thr Asn Asp Glu
                       - #    470             - #    475             - #
    480
            Lys Leu Ser Ser Asp Asn Ser - #Pro Pro Cys Leu Leu Pro Gly Trp
                       - #    485             - #    490             - #
    495
            Asp Ser Cys Asp Glu Ser Gln - #Ser Pro Pro Ile Glu Asn Asp Glu
                       - #    500             - #    505             - #
    510
            Trp Leu Ala Phe Leu His Arg - #Ser Ile Gln Glu Leu Leu Asp Gly
                       - #    515             - #    520             - #
    525
            Glu Phe Asp Ser Leu Lys Gln - #His Asn Leu Val Ser Ile Ile Val
                       - #    530             - #    535             - #
    540
            Ala Pro Leu Arg Asn Ser Lys - #Ala Ile Pro Lys Val Leu Gln Ser
                       - #    545             - #    550             - #
    555
            Val Ala Gln Leu Leu Ser Leu - #Pro Phe Val Leu Ala Glu Gln His
                       - #    560             - #    565             - #
    570
            Leu Val Ala Glu Ala Ile Lys - #Gly Val Tyr Ile Asp Val Lys Leu
                       - #    575             - #    580             - #
    585
            Val Pro Asn Leu Met Tyr Ala - #Cys Lys Leu Leu Leu Ser Gln Arg
                       - #    590             - #    595             - #
    600
            His Leu Thr Asp Ser Ala Ala - #Ser Leu Pro Ala Gly Thr Gly Val
                       - #    605             - #    610             - #
    615
            Ser Leu Ser Arg Thr Val Arg - #Ser Cys Ser Asp Leu Ser Ala Glu
                       - #    620             - #    625             - #
    630
            Glu Met Ser Thr Ala Cys Ser - #Leu Tyr Glu Leu Val Cys His Leu
                       - #    635             - #    640             - #
    645
            Val His Gln Gln Gln Gln Phe - #Leu Thr Gln Phe Cys Asp Ala Val
                       - #    650             - #    655             - #
    660
            Ala Ile Leu Ala Val Asn Asp - #Met Phe Ile Asn Phe Leu Thr His
                       - #    665             - #    670             - #
    675
            Asp Phe Lys Asp Ser Arg Pro - #Val Arg Leu Ala Ser Cys Met Leu
                       - #    680             - #    685             - #
    690
            Ala Leu Phe Cys Cys Val Leu - #Arg Glu Leu Pro Glu Asn Ala Glu
                       - #    695             - #    700             - #
    705
            Leu Val Glu Lys Ile Val Phe - #Asp Ser Arg Leu Gln Leu Ala Val
                       - #    710             - #    715             - #
    720
            Leu Leu Gln Ser Arg His His - #Leu Leu Arg Gln Arg Ala Cys Gln
                       - #    725             - #    730             - #
    735
            Met Leu Leu Leu Leu Ala Arg - #Phe Ser Leu Arg Gly Val Gln Cys
                       - #    740             - #    745             - #
    750
            Ile Trp Ser Gly Glu Leu Lys - #Ser Ala Leu Gln Ala Trp Pro Met
                       - #    755             - #    760             - #
    765
            Gln Gln Thr Cys Gln Ser Leu - #Arg Leu Glu Ala Ala Gln Thr Leu
                       - #    770             - #    775             - #
    780
            Asp Glu Leu Ser Gln Phe Ser - #Phe Phe Val Ala Gln Ala Thr Ala
                       - #    785             - #    790             - #
    795
       - -  - - <210> SEQ ID NO 24
      <211> LENGTH: 260
      <212> TYPE: PRT
      <213> ORGANISM: Homo sapiens
       - - <400> SEQUENCE: 24
       - -       Met Glu Lys Tyr His Val Leu - #Glu Met Ile Gly Glu Gly Ser
    Phe
              1          - #     5             - #     10             - #
    15
           Gly Arg Val Tyr Lys Gly Arg - #Arg Lys Tyr Ser Ala Gln Val Val
                       - #     20             - #     25             - #
    30
            Ala Leu Lys Phe Ile Pro Lys - #Leu Gly Arg Ser Glu Lys Glu Leu
                       - #     35             - #     40             - #
    45
            Arg Asn Leu Gln Arg Glu Ile - #Glu Ile Met Arg Gly Leu Arg His
                       - #     50             - #     55             - #
    60
            Pro Asn Ile Val His Met Leu - #Asp Ser Phe Glu Thr Asp Lys Glu
                       - #     65             - #     70             - #
    75
            Val Val Val Val Thr Asp Tyr - #Ala Glu Gly Glu Leu Phe Gln Ile
                       - #     80             - #     85             - #
    90
            Leu Glu Asp Asp Gly Lys Leu - #Pro Glu Asp Gln Val Gln Ala Ile
                       - #     95             - #    100             - #
    105
            Ala Ala Gln Leu Val Ser Ala - #Leu Tyr Tyr Leu His Ser His Arg
                       - #    110             - #    115             - #
    120
            Ile Leu His Arg Asp Met Lys - #Pro Gln Asn Ile Leu Leu Ala Lys
                       - #    125             - #    130             - #
    135
            Gly Gly Gly Ile Lys Leu Cys - #Asp Phe Gly Phe Ala Arg Ala Met
                       - #    140             - #    145             - #
    150
            Ser Thr Asn Thr Met Val Leu - #Thr Ser Ile Lys Gly Thr Pro Leu
                       - #    155             - #    160             - #
    165
            Tyr Met Ser Pro Glu Leu Val - #Glu Glu Arg Pro Tyr Asp His Thr
                       - #    170             - #    175             - #
    180
            Ala Asp Leu Trp Ser Val Gly - #Cys Ile Leu Tyr Glu Leu Ala Val
                       - #    185             - #    190             - #
    195
            Gly Thr Pro Pro Phe Tyr Ala - #Thr Ser Ile Phe Gln Leu Val Ser
                       - #    200             - #    205             - #
    210
            Leu Ile Leu Lys Asp Pro Val - #Arg Trp Pro Ser Thr Ile Ser Pro
                       - #    215             - #    220             - #
    225
            Cys Phe Lys Asn Phe Leu Gln - #Gly Leu Leu Thr Lys Asp Pro Arg
                       - #    230             - #    235             - #
    240
            Gln Arg Leu Ser Trp Pro Asp - #Leu Leu Tyr His Pro Phe Ile Ala
                       - #    245             - #    250             - #
    255
            Gly His Val Thr Ile
                        - #    260
       - -  - - <210> SEQ ID NO 25
      <211> LENGTH: 1315
      <212> TYPE: PRT
      <213> ORGANISM: artificial sequence
      <220> FEATURE:
      <223> OTHER INFORMATION: artificial sequence
       - - <400> SEQUENCE: 25
       - -       Met Glu Lys Tyr His Val Leu - #Glu Met Ile Gly Glu Gly Ser
    Phe
              1          - #     5             - #     10             - #
    15
           Gly Arg Val Tyr Lys Gly Arg - #Arg Lys Tyr Ser Ala Gln Val Val
                       - #     20             - #     25             - #
    30
            Ala Leu Arg Phe Ile Pro Lys - #Leu Gly Arg Ser Glu Lys Glu Leu
                       - #     35             - #     40             - #
    45
            Arg Asn Leu Gln Arg Glu Ile - #Glu Ile Met Arg Gly Leu Arg His
                       - #     50             - #     55             - #
    60
            Pro Asn Ile Val His Met Leu - #Asp Ser Phe Glu Thr Asp Lys Glu
                       - #     65             - #     70             - #
    75



            Val Val Val Val Thr Asp Tyr - #Ala Glu Gly Glu Leu Phe Gln Ile
                       - #     80             - #     85             - #
    90
            Leu Glu Asp Asp Gly Lys Leu - #Pro Glu Asp Gln Val Gln Ala Ile
                       - #     95             - #    100             - #
    105
            Ala Ala Gln Leu Val Ser Ala - #Leu Tyr Tyr Leu His Ser His Arg
                       - #    110             - #    115             - #
    120
            Ile Leu His Arg Asp Met Lys - #Pro Gln Asn Ile Leu Leu Ala Lys
                       - #    125             - #    130             - #
    135
            Gly Gly Gly Ile Lys Leu Cys - #Asp Phe Gly Phe Ala Arg Ala Met
                       - #    140             - #    145             - #
    150
            Ser Thr Asn Thr Met Val Leu - #Thr Ser Ile Lys Gly Thr Pro Leu
                       - #    155             - #    160             - #
    165
            Tyr Met Ser Pro Glu Leu Val - #Glu Glu Arg Pro Tyr Asp His Thr
                       - #    170             - #    175             - #
    180
            Ala Asp Leu Trp Ser Val Gly - #Cys Ile Leu Tyr Glu Leu Ala Val
                       - #    185             - #    190             - #
    195
            Gly Thr Pro Pro Phe Tyr Ala - #Thr Ser Ile Phe Gln Leu Val Ser
                       - #    200             - #    205             - #
    210
            Leu Ile Leu Lys Asp Pro Val - #Arg Trp Pro Ser Thr Ile Ser Pro
                       - #    215             - #    220             - #
    225
            Cys Phe Lys Asn Phe Leu Gln - #Gly Leu Leu Thr Lys Asp Pro Arg
                       - #    230             - #    235             - #
    240
            Gln Arg Leu Ser Trp Pro Asp - #Leu Leu Tyr His Pro Phe Ile Ala
                       - #    245             - #    250             - #
    255
            Gly His Val Thr Ile Ile Thr - #Glu Pro Ala Gly Pro Asp Leu Gly
                       - #    260             - #    265             - #
    270
            Thr Pro Phe Thr Ser Arg Leu - #Pro Pro Glu Leu Gln Val Leu Lys
                       - #    275             - #    280             - #
    285
            Asp Glu Gln Ala His Arg Leu - #Ala Pro Lys Gly Asn Gln Ser Arg
                       - #    290             - #    295             - #
    300
            Ile Leu Thr Gln Ala Tyr Lys - #Arg Met Ala Glu Glu Ala Met Gln
                       - #    305             - #    310             - #
    315
            Lys Lys His Gln Asn Thr Gly - #Pro Ala Leu Glu Gln Glu Asp Lys
                       - #    320             - #    325             - #
    330
            Thr Ser Lys Val Ala Pro Gly - #Thr Ala Pro Leu Pro Arg Leu Gly
                       - #    335             - #    340             - #
    345
            Ala Thr Pro Gln Glu Ser Ser - #Leu Leu Ala Gly Ile Leu Ala Ser
                       - #    350             - #    355             - #
    360
            Glu Leu Lys Ser Ser Trp Ala - #Lys Ser Gly Thr Gly Glu Val Pro
                       - #    365             - #    370             - #
    375
            Ser Ala Pro Arg Glu Asn Arg - #Thr Thr Pro Asp Cys Glu Arg Ala
                       - #    380             - #    385             - #
    390
            Phe Pro Glu Glu Arg Pro Glu - #Val Leu Gly Gln Arg Ser Thr Asp
                       - #    395             - #    400             - #
    405
            Val Val Asp Leu Glu Asn Glu - #Glu Pro Asp Ser Asp Asn Glu Trp
                       - #    410             - #    415             - #
    420
            Gln His Leu Leu Glu Thr Thr - #Glu Pro Val Pro Ile Gln Leu Lys
                       - #    425             - #    430             - #
    435
            Ala Pro Leu Thr Leu Leu Cys - #Asn Pro Asp Phe Cys Gln Arg Ile
                       - #    440             - #    445             - #
    450
            Gln Ser Gln Leu His Glu Ala - #Gly Gly Gln Ile Leu Lys Gly Ile
                       - #    455             - #    460             - #
    465
            Leu Glu Gly Ala Ser His Ile - #Leu Pro Ala Phe Arg Val Leu Ser
                       - #    470             - #    475             - #
    480
            Ser Leu Leu Ser Ser Cys Ser - #Asp Ser Val Ala Leu Tyr Ser Phe
                       - #    485             - #    490             - #
    495
            Cys Arg Glu Ala Gly Leu Pro - #Gly Leu Leu Leu Ser Leu Leu Arg
                       - #    500             - #    505             - #
    510
            His Ser Gln Glu Ser Asn Ser - #Leu Gln Gln Gln Ser Trp Tyr Gly
                       - #    515             - #    520             - #
    525
            Thr Phe Leu Gln Asp Leu Met - #Ala Val Ile Gln Ala Tyr Phe Ala
                       - #    530             - #    535             - #
    540
            Cys Thr Phe Asn Leu Glu Arg - #Ser Gln Thr Ser Asp Ser Leu Gln
                       - #    545             - #    550             - #
    555
            Val Phe Gln Glu Ala Ala Asn - #Leu Phe Leu Asp Leu Leu Gly Lys
                       - #    560             - #    565             - #
    570
            Leu Leu Ala Gln Pro Asp Asp - #Ser Glu Gln Thr Leu Arg Arg Asp
                       - #    575             - #    580             - #
    585
            Ser Leu Met Cys Phe Thr Val - #Leu Cys Glu Ala Met Asp Gly Asn
                       - #    590             - #    595             - #
    600
            Ser Arg Ala Ile Ser Lys Ala - #Phe Tyr Ser Ser Leu Leu Thr Thr
                       - #    605             - #    610             - #
    615
            Gln Gln Val Val Leu Asp Gly - #Leu Leu His Gly Leu Thr Val Pro
                       - #    620             - #    625             - #
    630
            Gln Leu Pro Val His Thr Pro - #Gln Gly Ala Pro Gln Val Ser Gln
                       - #    635             - #    640             - #
    645
            Pro Leu Arg Glu Gln Ser Glu - #Asp Ile Pro Gly Ala Ile Ser Ser
                       - #    650             - #    655             - #
    660
            Ala Leu Ala Ala Ile Cys Thr - #Ala Pro Val Gly Leu Pro Asp Cys
                       - #    665             - #    670             - #
    675
            Trp Asp Ala Lys Glu Gln Val - #Cys Trp His Leu Ala Asn Gln Leu
                       - #    680             - #    685             - #
    690
            Thr Glu Asp Ser Ser Gln Leu - #Arg Pro Ser Leu Ile Ser Gly Leu
                       - #    695             - #    700             - #
    705
            Gln His Pro Ile Leu Cys Leu - #His Leu Leu Lys Val Leu Tyr Ser
                       - #    710             - #    715             - #
    720
            Cys Cys Leu Val Ser Glu Gly - #Leu Cys Arg Leu Leu Gly Gln Glu
                       - #    725             - #    730             - #
    735
            Pro Leu Ala Leu Glu Ser Leu - #Phe Met Leu Ile Gln Gly Lys Val
                       - #    740             - #    745             - #
    750
            Lys Val Val Asp Trp Glu Glu - #Ser Thr Glu Val Thr Leu Tyr Phe
                       - #    755             - #    760             - #
    765
            Leu Ser Leu Leu Val Phe Arg - #Leu Gln Asn Leu Pro Cys Gly Met
                       - #    770             - #    775             - #
    780
            Glu Lys Leu Gly Ser Asp Val - #Ala Thr Leu Phe Thr His Ser His
                       - #    785             - #    790             - #
    795
            Val Val Ser Leu Val Ser Ala - #Ala Ala Cys Leu Leu Gly Gln Leu
                       - #    800             - #    805             - #
    810
            Gly Gln Gln Gly Val Thr Phe - #Asp Leu Gln Pro Met Glu Trp Met
                       - #    815             - #    820             - #
    825
            Ala Ala Ala Thr His Ala Leu - #Ser Ala Pro Ala Glu Val Arg Leu
                       - #    830             - #    835             - #
    840
            Thr Pro Pro Gly Ser Cys Gly - #Phe Tyr Asp Gly Leu Leu Ile Leu
                       - #    845             - #    850             - #
    855
            Leu Leu Gln Leu Leu Thr Glu - #Gln Gly Lys Ala Ser Leu Ile Arg
                       - #    860             - #    865             - #
    870
            Asp Met Ser Ser Ser Glu Met - #Trp Thr Val Leu Trp His Arg Phe
                       - #    875             - #    880             - #
    885
            Ser Met Val Leu Arg Leu Pro - #Glu Glu Ala Ser Ala Gln Glu Gly
                       - #    890             - #    895             - #
    900
            Glu Leu Ser Leu Ser Ser Pro - #Pro Ser Pro Glu Pro Asp Trp Thr
                       - #    905             - #    910             - #
    915
            Leu Ile Ser Pro Gln Gly Met - #Ala Ala Leu Leu Ser Leu Ala Met
                       - #    920             - #    925             - #
    930
            Ala Thr Phe Thr Gln Glu Pro - #Gln Leu Cys Leu Ser Cys Leu Ser
                       - #    935             - #    940             - #
    945
            Gln His Gly Ser Ile Leu Met - #Ser Ile Leu Lys His Leu Leu Cys
                       - #    950             - #    955             - #
    960
            Pro Ser Phe Leu Asn Gln Leu - #Arg Gln Ala Pro His Gly Ser Glu
                       - #    965             - #    970             - #
    975
            Phe Leu Pro Val Val Val Leu - #Ser Val Cys Gln Leu Leu Cys Phe
                       - #    980             - #    985             - #
    990
            Pro Phe Ala Leu Asp Met Asp - #Ala Asp Leu Leu Ile Val Val Leu
                       - #    995             - #   1000              - #
    1005
            Ala Asp Leu Arg Asp Ser Glu - #Val Ala Ala His Leu Leu Gln Val
                       - #   1010              - #  1015               - #
    1020
            Cys Cys Tyr His Leu Pro Leu - #Met Gln Val Glu Leu Pro Ile Ser
                       - #   1025              - #  1030               - #
    1035
            Leu Leu Thr Arg Leu Ala Leu - #Met Asp Pro Thr Ser Leu Asn Gln
                       - #   1040              - #  1045               - #
    1050
            Phe Val Asn Thr Val Ser Ala - #Ser Pro Arg Thr Ile Val Ser Phe
                       - #   1055              - #  1060               - #
    1065
            Leu Ser Val Ala Leu Leu Ser - #Asp Gln Pro Leu Leu Thr Ser Asp
                       - #   1070              - #  1075               - #
    1080
            Leu Leu Ser Leu Leu Ala His - #Thr Ala Arg Val Leu Ser Pro Ser
                       - #   1085              - #  1090               - #
    1095
            His Leu Ser Phe Ile Gln Glu - #Leu Leu Ala Gly Ser Asp Glu Ser
                       - #   1100              - #  1105               - #
    1110
            Tyr Arg Pro Leu Arg Ser Leu - #Leu Gly His Pro Glu Asn Ser Val
                       - #   1115              - #  1120               - #
    1125
            Arg Ala His Thr Tyr Arg Leu - #Leu Gly His Leu Leu Gln His Ser
                       - #   1130              - #  1135               - #
    1140
            Met Ala Leu Arg Gly Ala Leu - #Gln Ser Gln Ser Gly Leu Leu Ser
                       - #   1145              - #  1150               - #
    1155
            Leu Leu Leu Leu Gly Leu Gly - #Asp Lys Asp Pro Val Val Arg Cys
                       - #   1160              - #  1165               - #
    1170
            Ser Ala Ser Phe Ala Val Gly - #Asn Ala Ala Tyr Gln Ala Gly Pro
                       - #   1175              - #  1180               - #
    1185
            Leu Gly Pro Ala Leu Ala Ala - #Ala Val Pro Ser Met Thr Gln Leu
                       - #   1190              - #  1195               - #
    1200
            Leu Gly Asp Pro Gln Ala Gly - #Ile Arg Arg Asn Val Ala Ser Ala
                       - #   1205              - #  1210               - #
    1215
            Leu Gly Asn Leu Gly Pro Glu - #Gly Leu Gly Glu Glu Leu Leu Gln
                       - #   1220              - #  1225               - #
    1230
            Cys Glu Val Pro Gln Arg Leu - #Leu Glu Met Ala Cys Gly Asp Pro
                       - #   1235              - #  1240               - #
    1245
            Gln Pro Asn Val Lys Glu Ala - #Ala Leu Ile Ala Leu Arg Ser Leu
                       - #   1250              - #  1255               - #
    1260
            Gln Gln Glu Pro Gly Ile His - #Gln Val Leu Val Ser Leu Gly Ala
                       - #   1265              - #  1270               - #
    1275
            Ser Glu Lys Leu Ser Leu Leu - #Ser Leu Gly Asn Gln Ser Leu Pro
                       - #   1280              - #  1285               - #
    1290
            His Ser Ser Pro Arg Pro Ala - #Ser Ala Lys His Cys Arg Lys Leu
                       - #   1295              - #  1300               - #
    1305
            Ile His Leu Leu Arg Pro Ala - #His Ser Met
                        - #   1310              - #  1315
       - -  - - <210> SEQ ID NO 26
      <211> LENGTH: 4586
      <212> TYPE: DNA
      <213> ORGANISM: Drosophila virilis
       - - <400> SEQUENCE: 26
       - -       tgcagagtct gggccatcgg ctagctctgt agatgt - #gtaa tagaggcatc
    50



            ttcgcgcgca gcatcgattc gcgctccagt tggttg - #agat gccgaatgtt 100
           ggtgcgcgtc tcaaagatga cgccgacatg gtgcat - #atac agaaaaaaga 150
           aacccgagcc taatggccgc gtattatcgc tgaggc - #ggcc ggcgattttc 200
           aacaaatgct acttaccaat tagcgcgtgc gataaa - #atta cgtacaaatt 250
           ggcgtcgcgc atatttctgg cgtttgtgtt gcgtgt - #atct agttagtggg 300
           ctcgtctatt cattatttac ttttggggcg tctgtt - #tgat caaattagca 350
           gtgtctccta tgatatgcct gcagctttta cccgta - #aaca aaattatttg 400
           ccacagctga tttattcgtt gccatgtaga ttaatc - #agct gtttcgcaat 450
           ttttaaaacc aggtgacttt ttaaaattgt accagc - #tgtg tgtatcgatg 500
           tgcaagcata ctcattacgc catatgctgg tatatt - #tata tcgaatataa 550
           acggttttgg tattttaata atcttaaaga agaaat - #agtt atgtgctgtg 600
           tatatgttca tcaagaactg ttcaaaatgt gcgcca - #tact gatgttaatt 650
           ttgttttgct ggtttttttt gggaaaataa attgac - #gtgt tgatgtctcc 700
           gaatatatcg atacaatagc tatcattcgg acaaga - #tatc gatatgtgga 750
           gtgtgttcgg tattttgcct ttagtttttt gttttt - #aaat tgcagtcaca 800
           ctgcggctta ttgaatttaa ggcacttcaa agcgca - #tttt actgtagaaa 850
           gttgagttct atttgcggtg acaatggacc gctacg - #cggt tagctctctg 900
           gtaggacaag gctcatttgg ctgtgtgtac aaggcc - #cagc ggcgcgatga 950
           tgacaaagtg gtggccatca aagtcatatc aaaggt - #gagc tcaattgcat 1000
           cccggcttag ctgaataaaa gagtattcta cgaatt - #ggcg tgttctttgt 1050
           ttgcagcgtg gtcgttccaa tcgcgagctt aagaac - #ctgc gtcgcgaatg 1100
           tgacattcag gcgcgtctca agcatccgca tgttat - #agaa atggtggagt 1150
           cgttcgaatc caaattcgat ttgttcgtgg tcaccg - #agtt cgctctaatg 1200
           gacttgcatc gatatttgtc ctttaatggc gccatg - #cccg aggagcacgc 1250
           acagcgtgtt gtctgtcatt tggtgtcggc gctcta - #ttat ctgcactcga 1300
           atcgcatact gcatcgggat ctaaagccgc aaaatg - #tgct gttggacaaa 1350
           aacatgcacg ccaagctctg cgactttggg ctggca - #cgca acatgacgat 1400
           gggcacacat gtgttgactt ccataaaggg cacgcc - #gctt tatatggcgc 1450
           cggagctgct ggctgagcag ccgtacgatc accagg - #caga tatgtggtcg 1500
           ctgggatgca ttgcctatga gagtatggcg ggccag - #ccgc cgttctgcgc 1550
           aacctctata ctgcatctgg tgaagctgat caagca - #cgag gacgtcaaat 1600
           ggccgagcac gctgagcagc gagtgccgtt cctttt - #tgca gggcttgctc 1650
           gagaaggatc ctagcatgcg catctcatgg acgcag - #ctgc tttgccatcc 1700
           ttttgtcgag ggcaagctat acatagccga ggtaca - #ggca gcacaaactt 1750
           cgccctttat aaatccccag ctggccaagg acacca - #aaaa atcacagcaa 1800
           ttgaggtgcg tttataacgt gtactgtagc cagctc - #cact tatcgttcaa 1850
           tttttatgta ggcatgtagg cgcagatttg ggcgat - #gtct tggcagcgtt 1900
           aaagttgagc gatgtggcca atgaaaactt gagcac - #atcg cgagatagta 1950
           tcaatgccat tgcgccgagt gacattgagc agctgg - #aaac cgatgttgag 2000
           gataatgtgc atcggcttat agtgccattt gcagat - #attt cctacagaga 2050
           gttgccatgc ggcactgcag cagctgctcg tcgagc - #tggt gccatgccac 2100
           tgattaattc gcaaacctgc tttgtaagtg gcaact - #ccaa tatgatactc 2150
           aatcatctga acgacaattt tgcaatcgaa gcgcct - #gctt cgagcgcaac 2200
           caagtccatg aagtcgaagc tgaagctggc tctcaa - #tata aaacagtcgc 2250
           gtagcaagga tttggaaaag cgtaagctga gtcaaa - #attt ggataacttt 2300
           tcgctgcgcc tgggacagag cattgacata gaagtg - #cagc gcaaaacaac 2350
           tgagatgctc acgcagcaat cgcaggcaca acagct - #gcag gataggaaga 2400
           cacagcagct gaagcaatcg atgcattcca ccaacg - #acga gaaattgagc 2450
           agcgagtgag taaatgcatc catatttaaa agtgaa - #gctc tctaaagcta 2500
           tttggtttat aatagcaatt cgccgccttg tctgtt - #gccc ggttgggaca 2550
           gctgcgatga atctcagagc ccgcccattg agaatg - #acga gtggctggcg 2600
           ttcttgcatc gctccataca ggagctgctg gacggc - #gaat ttgattcgct 2650
           gaagcagcac aatctagtca gcataattgt ggcgcc - #attg cgaaactcca 2700
           aggccatacc caaggtgctg cagagcgtgg cgcagc - #tgct gtcgctgccc 2750
           tttgtgctgg ccgaacagca tttggtagcg gaggcc - #ataa aaggagttta 2800
           tattgatgtc aagctggtgc ccaacttaat gtacgc - #ctgc aagctgcttc 2850
           tctcgcagcg ccaccttacc gattcggctg cttcac - #tgcc agccggcacg 2900
           ggcgtctccc tgagtcgaac cgtacgcagc tgctcc - #gacc tgagtgccga 2950
           ggagatgagc accgcctgca gcctgtacga gctggt - #ctgc catctggtcc 3000
           atcagcagca gcagttcctc acccagttct gtgacg - #ctgt ggcaatactc 3050
           gccgtcaacg acatgttcat aaattttctt acacat - #ggtg agcagctggc 3100
           tggacacagt gtgagacgca agcttaacca ttcctt - #gctt tgcagatttt 3150
           aaggatagca ggccggtgcg actcgctagc tgcatg - #ctgg cattgttctg 3200
           ttgcgttttg cgtgaactac ccgagaacgc cgagct - #ggtg gagaaaattg 3250
           tatttgactc gcgcctacag ctggccgtcc tgctgc - #agag ccgtcatcat 3300
           ttgttgcgtc agcgcgcctg tcaaatgctg ttgcta - #ttgg cacgctttag 3350
           cctgcgcggc gtacagtgca tctggagtgg ggagct - #gaag agtgcgctcc 3400
           aggcgtggcc gatgcagcaa acgtgtcaat cattgc - #gact ggaagccgcc 3450
           caaacgctgg atgagcttag ccagttcagc ttcttt - #gttg ctcaggcaac 3500
           tgcttagtct ttattaataa ttgtacttgt atttgt - #ttaa taaatcttaa 3550
           tccttgtcta gccgaacaga ccttccaaat tgcctt - #gaaa gtagtcgagc 3600
           agctcgtcca gatagctgct aaagccatca aagccc - #aaaa ggtagctacc 3650
           attacagtcc tgctcgtaca tctcgtttag tttcga - #aata tccttatccg 3700
           acagccgcgc gctggcccag tgaggcatac ggatcc - #taat gataatagca 3750
           tgcattatta ttatttttca caatgtgtta ttcgtt - #taat acttataaaa 3800
           ccttaaattg tatgcatgta tgtatctatc ttatac - #ctaa ttaatgaatg 3850
           aaatttatta acttgtctat ggatgtatgt gcatgt - #atgt atgtatgtat 3900
           gtatgcataa aaatgtatgt tcatttataa caaacg - #caga caaagataac 3950
           gatctgctgc tctacttccc gaatctcata aattca - #agta cgccccgcag 4000
           atttcacgag tacatcacaa gtgttttttt ttaaca - #agta atgttggtat 4050
           gtatttatgt atatatgtat ttaagtatgt atgtat - #ttat gtatgtatgt 4100
           atgtatttat gtatgtatgt atttatgtat gtatgt - #attt atgtatgtat 4150
           gtatttatgt atgtatttat ttatgtatat atgtat - #ttaa gtatgtatgt 4200
           atgtatttat gcatttatgt atttatgtat gtatgt - #ataa gagtatgtgt 4250
           gtgtgtagat acatgtatgt atgtatgtat gtatgc - #gtgt atatttattt 4300
           atagtaaaca taccaacttt acttcccgct gccttg - #cgaa tttaaaataa 4350
           cgtattttta aatgatgccc tactcctcga ttctca - #aaca tttaagtaag 4400
           ctctacaggt ttttccgatt tgattgtttt gtaaag - #ttgt gttttttttt 4450
           ctgctcgatc tcttgtgtat tctctactct ttgtgt - #gcct ctctttagtt 4500
           ttctctcctt ctctcttgct ctcccctgtt ctctct - #ctat ctctctccct 4550
           ccctctttcc acctatctca ttctctttct aagctt - # 4586
      - -  - - <210> SEQ ID NO 27
     <211> LENGTH: 1055
     <212> TYPE: PRT
     <213> ORGANISM: Homo sapiens
      - - <400> SEQUENCE: 27
      - -       Ile Thr Glu Pro Ala Gly Pro - #Asp Leu Gly Thr Pro Phe Thr
    Ser
              1          - #     5             - #     10             - #
    15
           Arg Leu Pro Pro Glu Leu Gln - #Val Leu Lys Asp Glu Gln Ala His
                       - #     20             - #     25             - #
    30
            Arg Leu Ala Pro Lys Gly Asn - #Gln Ser Arg Ile Leu Thr Gln Ala
                       - #     35             - #     40             - #
    45
            Tyr Lys Arg Met Ala Glu Glu - #Ala Met Gln Lys Lys His Gln Asn
                       - #     50             - #     55             - #
    60
            Thr Gly Pro Ala Leu Glu Gln - #Glu Asp Lys Thr Ser Lys Val Ala
                       - #     65             - #     70             - #
    75
            Pro Gly Thr Ala Pro Leu Pro - #Arg Leu Gly Ala Thr Pro Gln Glu
                       - #     80             - #     85             - #
    90
            Ser Ser Leu Leu Ala Gly Ile - #Leu Ala Ser Glu Leu Lys Ser Ser
                       - #     95             - #    100             - #
    105
            Trp Ala Lys Ser Gly Thr Gly - #Glu Val Pro Ser Ala Pro Arg Glu
                       - #    110             - #    115             - #
    120
            Asn Arg Thr Thr Pro Asp Cys - #Glu Arg Ala Phe Pro Glu Glu Arg
                       - #    125             - #    130             - #
    135
            Pro Glu Val Leu Gly Gln Arg - #Ser Thr Asp Val Val Asp Leu Glu
                       - #    140             - #    145             - #
    150
            Asn Glu Glu Pro Asp Ser Asp - #Asn Glu Trp Gln His Leu Leu Glu
                       - #    155             - #    160             - #
    165
            Thr Thr Glu Pro Val Pro Ile - #Gln Leu Lys Ala Pro Leu Thr Leu
                       - #    170             - #    175             - #
    180
            Leu Cys Asn Pro Asp Phe Cys - #Gln Arg Ile Gln Ser Gln Leu His
                       - #    185             - #    190             - #
    195
            Glu Ala Gly Gly Gln Ile Leu - #Lys Gly Ile Leu Glu Gly Ala Ser
                       - #    200             - #    205             - #
    210
            His Ile Leu Pro Ala Phe Arg - #Val Leu Ser Ser Leu Leu Ser Ser
                       - #    215             - #    220             - #
    225
            Cys Ser Asp Ser Val Ala Leu - #Tyr Ser Phe Cys Arg Glu Ala Gly
                       - #    230             - #    235             - #
    240
            Leu Pro Gly Leu Leu Leu Ser - #Leu Leu Arg His Ser Gln Glu Ser
                       - #    245             - #    250             - #
    255
            Asn Ser Leu Gln Gln Gln Ser - #Trp Tyr Gly Thr Phe Leu Gln Asp
                       - #    260             - #    265             - #
    270
            Leu Met Ala Val Ile Gln Ala - #Tyr Phe Ala Cys Thr Phe Asn Leu
                       - #    275             - #    280             - #
    285
            Glu Arg Ser Gln Thr Ser Asp - #Ser Leu Gln Val Phe Gln Glu Ala
                       - #    290             - #    295             - #
    300
            Ala Asn Leu Phe Leu Asp Leu - #Leu Gly Lys Leu Leu Ala Gln Pro
                       - #    305             - #    310             - #
    315
            Asp Asp Ser Glu Gln Thr Leu - #Arg Arg Asp Ser Leu Met Cys Phe
                       - #    320             - #    325             - #
    330
            Thr Val Leu Cys Glu Ala Met - #Asp Gly Asn Ser Arg Ala Ile Ser
                       - #    335             - #    340             - #
    345
            Lys Ala Phe Tyr Ser Ser Leu - #Leu Thr Thr Gln Gln Val Val Leu
                       - #    350             - #    355             - #
    360
            Asp Gly Leu Leu His Gly Leu - #Thr Val Pro Gln Leu Pro Val His
                       - #    365             - #    370             - #
    375
            Thr Pro Gln Gly Ala Pro Gln - #Val Ser Gln Pro Leu Arg Glu Gln
                       - #    380             - #    385             - #
    390
            Ser Glu Asp Ile Pro Gly Ala - #Ile Ser Ser Ala Leu Ala Ala Ile
                       - #    395             - #    400             - #
    405
            Cys Thr Ala Pro Val Gly Leu - #Pro Asp Cys Trp Asp Ala Lys Glu
                       - #    410             - #    415             - #
    420
            Gln Val Cys Trp His Leu Ala - #Asn Gln Leu Thr Glu Asp Ser Ser
                       - #    425             - #    430             - #
    435
            Gln Leu Arg Pro Ser Leu Ile - #Ser Gly Leu Gln His Pro Ile Leu
                       - #    440             - #    445             - #
    450
            Cys Leu His Leu Leu Lys Val - #Leu Tyr Ser Cys Cys Leu Val Ser
                       - #    455             - #    460             - #
    465
            Glu Gly Leu Cys Arg Leu Leu - #Gly Gln Glu Pro Leu Ala Leu Glu
                       - #    470             - #    475             - #
    480
            Ser Leu Phe Met Leu Ile Gln - #Gly Lys Val Lys Val Val Asp Trp
                       - #    485             - #    490             - #
    495
            Glu Glu Ser Thr Glu Val Thr - #Leu Tyr Phe Leu Ser Leu Leu Val
                       - #    500             - #    505             - #
    510
            Phe Arg Leu Gln Asn Leu Pro - #Cys Gly Met Glu Lys Leu Gly Ser
                       - #    515             - #    520             - #
    525
            Asp Val Ala Thr Leu Phe Thr - #His Ser His Val Val Ser Leu Val
                       - #    530             - #    535             - #
    540
            Ser Ala Ala Ala Cys Leu Leu - #Gly Gln Leu Gly Gln Gln Gly Val
                       - #    545             - #    550             - #
    555
            Thr Phe Asp Leu Gln Pro Met - #Glu Trp Met Ala Ala Ala Thr His
                       - #    560             - #    565             - #
    570
            Ala Leu Ser Ala Pro Ala Glu - #Val Arg Leu Thr Pro Pro Gly Ser
                       - #    575             - #    580             - #
    585
            Cys Gly Phe Tyr Asp Gly Leu - #Leu Ile Leu Leu Leu Gln Leu Leu
                       - #    590             - #    595             - #
    600
            Thr Glu Gln Gly Lys Ala Ser - #Leu Ile Arg Asp Met Ser Ser Ser
                       - #    605             - #    610             - #
    615
            Glu Met Trp Thr Val Leu Trp - #His Arg Phe Ser Met Val Leu Arg
                       - #    620             - #    625             - #
    630
            Leu Pro Glu Glu Ala Ser Ala - #Gln Glu Gly Glu Leu Ser Leu Ser
                       - #    635             - #    640             - #
    645
            Ser Pro Pro Ser Pro Glu Pro - #Asp Trp Thr Leu Ile Ser Pro Gln
                       - #    650             - #    655             - #
    660
            Gly Met Ala Ala Leu Leu Ser - #Leu Ala Met Ala Thr Phe Thr Gln
                       - #    665             - #    670             - #
    675
            Glu Pro Gln Leu Cys Leu Ser - #Cys Leu Ser Gln His Gly Ser Ile
                       - #    680             - #    685             - #
    690
            Leu Met Ser Ile Leu Lys His - #Leu Leu Cys Pro Ser Phe Leu Asn
                       - #    695             - #    700             - #
    705
            Gln Leu Arg Gln Ala Pro His - #Gly Ser Glu Phe Leu Pro Val Val
                       - #    710             - #    715             - #
    720
            Val Leu Ser Val Cys Gln Leu - #Leu Cys Phe Pro Phe Ala Leu Asp
                       - #    725             - #    730             - #
    735
            Met Asp Ala Asp Leu Leu Ile - #Val Val Leu Ala Asp Leu Arg Asp
                       - #    740             - #    745             - #
    750
            Ser Glu Val Ala Ala His Leu - #Leu Gln Val Cys Cys Tyr His Leu
                       - #    755             - #    760             - #
    765
            Pro Leu Met Gln Val Glu Leu - #Pro Ile Ser Leu Leu Thr Arg Leu
                       - #    770             - #    775             - #
    780
            Ala Leu Met Asp Pro Thr Ser - #Leu Asn Gln Phe Val Asn Thr Val
                       - #    785             - #    790             - #



    795
            Ser Ala Ser Pro Arg Thr Ile - #Val Ser Phe Leu Ser Val Ala Leu
                       - #    800             - #    805             - #
    810
            Leu Ser Asp Gln Pro Leu Leu - #Thr Ser Asp Leu Leu Ser Leu Leu
                       - #    815             - #    820             - #
    825
            Ala His Thr Ala Arg Val Leu - #Ser Pro Ser His Leu Ser Phe Ile
                       - #    830             - #    835             - #
    840
            Gln Glu Leu Leu Ala Gly Ser - #Asp Glu Ser Tyr Arg Pro Leu Arg
                       - #    845             - #    850             - #
    855
            Ser Leu Leu Gly His Pro Glu - #Asn Ser Val Arg Ala His Thr Tyr
                       - #    860             - #    865             - #
    870
            Arg Leu Leu Gly His Leu Leu - #Gln His Ser Met Ala Leu Arg Gly
                       - #    875             - #    880             - #
    885
            Ala Leu Gln Ser Gln Ser Gly - #Leu Leu Ser Leu Leu Leu Leu Gly
                       - #    890             - #    895             - #
    900
            Leu Gly Asp Lys Asp Pro Val - #Val Arg Cys Ser Ala Ser Phe Ala
                       - #    905             - #    910             - #
    915
            Val Gly Asn Ala Ala Tyr Gln - #Ala Gly Pro Leu Gly Pro Ala Leu
                       - #    920             - #    925             - #
    930
            Ala Ala Ala Val Pro Ser Met - #Thr Gln Leu Leu Gly Asp Pro Gln
                       - #    935             - #    940             - #
    945
            Ala Gly Ile Arg Arg Asn Val - #Ala Ser Ala Leu Gly Asn Leu Gly
                       - #    950             - #    955             - #
    960
            Pro Glu Gly Leu Gly Glu Glu - #Leu Leu Gln Cys Glu Val Pro Gln
                       - #    965             - #    970             - #
    975
            Arg Leu Leu Glu Met Ala Cys - #Gly Asp Pro Gln Pro Asn Val Lys
                       - #    980             - #    985             - #
    990
            Glu Ala Ala Leu Ile Ala Leu - #Arg Ser Leu Gln Gln Glu Pro Gly
                       - #    995             - #   1000              - #
    1005
            Ile His Gln Val Leu Val Ser - #Leu Gly Ala Ser Glu Lys Leu Ser
                       - #   1010              - #  1015               - #
    1020
            Leu Leu Ser Leu Gly Asn Gln - #Ser Leu Pro His Ser Ser Pro Arg
                       - #   1025              - #  1030               - #
    1035
            Pro Ala Ser Ala Lys His Cys - #Arg Lys Leu Ile His Leu Leu Arg
                       - #   1040              - #  1045               - #
    1050
            Pro Ala His Ser Met
                        - #   1055
    __________________________________________________________________________


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